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Quantitative and structural analysis of inhibitors of phospholipase A2
,Quantitative Michael University
and structural analysis of inhibitors ‘phospholipase A2 H. Gelb, Otto
Berg and Mahendra
of Washington, Seattle, Washington, and University of Delaware,
of
K. Jain
USA, Uppsala University, Newark, Delaware, USA
Uppsala,
Sweden
The evaluation of competitive inhibitors of phospholipase A, is best carried out by analysing the enzyme under conditions in which it binds irreversibly to the substrate vesicle and catalyses a processive reaction in which all of the phospholipid molecules on the outer layer of the vesicle are hydrolysed. Recent structural analyses of phospholipase AZ-inhibitor complexes suggest a mechanism for the catalytic site.
Current
Opinion
in Structural
Biology
1:836-843
zymes are thought to play a role in degenerate joint diseases. In addition, PLA2 inhibitors may block the liberation of arachidonic acid during the eicosanoid cascade. The scope of this review is aimed at recent developments in the kinetic and structural analysis of PLA2.
Introduction
Extracellular secretory phospholipase AZS (P&s) are small (14kD), water-soluble enzymes that hydrolyse the ester at the ~2-2 position of phospholipids. Abundant forms are the pancreatic P&s, which are involved in lipid digestion, and the enzymes found in a variety of animal venoms, which contribute to the toxicity of these substances. The properties of these enzymes have been reviewed [l-3]. More recently, PLA2s have been detected in a variety of different animal fluids and tissues and have been shown to be secreted from certain cells such as platelets. The role of these enzymes in inflammation and signal transduction is currently under intensive study [4]. All of these extracellular PLA2s require approximately 1O-4 M calcium for activity and contain multiple disulfide bonds.
Does phospholipase A, obey the Michaelis-Menten formalism?
The answer to this question is yes. This is reassuring because the Michaelis-Menten formalism has been useful in describing the quantitative behavior of essentially every known enzyme that operates in a homogeneous environment. Thus, rather than abandoning tradition, a serious effort should be made to use this basic paradigm of enzyme kinetics for the kinetic analysis of inter-facial catalysis by PIA*. The crux of the problem in achieving this is that the substrate is present in aggregates, dispersed as micelles or vesicles, which have a finite size. The kinetic analysis of any system requires a perfect mixing of all reactants so that every enzyme on average ‘sees’ the same environment of substrate and product at any particular time. In solution enzymology, rapid mixing is normally not a concern. When studying interfacial catalysis, however, special care has to be taken to ensure that all enzymes operate in a common environment.
A novel PI.& with a Mr -88 000 has been recently detected in the cytoplasm of macrophages [5-8], kidney [9] and platelets [lo]. In the presence of submicromolar amounts of calcium, this enzyme becomes catalytically active and is tmnslocated from the cytoplasm to the membrane of cells. This enzyme may serve to release arachidonic acid from the membrane pool for the biosynthesis of the eicosanoids. Because naturally occurring phospholipids are always present in interfaces, PLA$ have evolved to be considerably more active on substrate aggregates. The quantitative analysis of the interfacial catalysis by PLA2 is more challenging than that of enzyme systems that operate in a homogeneous aqueous environment. The inherent heterogeneity of the lipid-water interface often produces anomalous kinetic behavior which in turn has led to misconceptions about substrate preferences and inhibition with this class of enzymes. Inhibitors of interfacial catalysis by PIA;! may be medicinally significant as these en-
For example, consider the action of PI.& on a mixedmicellar substrate, a commonly studied system, in which the phospholipid molecules are dispersed in an ensemble of detergent micelles [ 1,2,11]. If there is no exchange of enzyme or lipid between these micelles, the reaction would quickly come to a halt after the 10 or so substrate molecules in a mixed micelle containing the enzyme have been hydrolysed. As a typical turnover number for a PIA2
Abbreviations 2H-GPC-2-hexadecyl-glycero-sn-3-phosphocholine;
DMPM-1,2-dimyristoyl-glycero-sn-3-phosphomethanol; 836
1991,
@
Current
Biology
Ltd
ISSN
0959440X
PL4*-phospholipase
A,.
Quantitative
and structural
analysis
of inhibitors
A, Celb, Berg and lain
of phospholipase
is well over lCOs- *, the contents of a single mixed-micelle containing PM, will be signiticantly altered in just a few milliseconds. If the lipolysis products are removed from the micelle to other miceUes by lipid exchange and the substrate is replenished by exchange from miceUes with no enzymes, the reaction can continue for an extended period. Only if these exchange processes are fast on the millisecond timescale will the observed steadystate enzymatic velocity be free of distortions resulting from low miceUar exchange rates, and will all of the micelles change in composition in a uniform way, with all enzyme molecules present in a common environment at any point in time. Alternatively, the enzyme could hop among the ensemble of miceUes. For there to be no distortion in enzyme velocity in this situation, the enzyme must leave the miceUe before it has depleted the substrate, i.e. the rate constant for the desorption of the PIA* from the miceUe must be larger than the rate constant for change in the composition of a single miceUe. Only in this fast hopping mode will all of the miceUes change in composition in a uniform way and will all enzyme molecules exist in a common environment. These requirements of either fast lipid or fast enzyme exchange are hard to verify experimentaUy. The only reported rates of phospholipid exchange in mixed miceUes come from recent studies by Nichols and coworkers [12**,13]. Here, the exchange rates of fluoresce&y labeled phospholipids in miceUes of bile salts were measured by a fluorescence dequenching approach. For long-chain phospholipids, which essentially never enter the aqueous phase, intermiceuar exchange occurs by a process that requires the collision of miceUes. With the concentrations of components that are typically used in PLA, experiments (510 mM detergent, l-2 mM phospholipids), the half-times for exchange range from m 100 ms to several seconds, depending on the structure of the bile salt. This is clearly not fast on the l-5ms timescale required for rapid averaging during PIAz turnover. The problem may be even more complicated as one reaUy needs to measure the rate of lipid exchange not in a pure mixed-miceUe but in one that contains a bound enzyme. One could imagine that a bound PLA2 might ‘trap’ lipid molecules that make up a protein-lipid complex. The rate constant for PIAz desorption from miceUes and vesicles has been measured by stopped-Uow fluorescence [ 141. An upper-limit value of about 4s-t was measured and found to be remarkably insensitive to the nature of the phospholipid interface. Thus, it appears that the desorption of enzyme from a miceUe may not be fast on the few millisecond timescale. In summary, one cannot safely conclude that exchange of components in miceUar enzymology will in all cases be fast enough to ensure that the intrinsic catalytic properties of the PLA2 are being measured. Rates of enzymatic turnover in miceUes may be more a reflection of the rates of exchange of lipid or enzyme among the ensemble of substrate aggregates. It is the problem of substrate aggregates of Unite size and the requirement for rapid mixing, that can lead to problems in the kinetic analysis of interfacial catalysis.
0
Experimentally 1. Enzyme 2. Substrate vesicles.
verified does
not
and
3. The vesicle:enzyme there is at most
constraints: exchange
products
one
do
(‘hop’) not
between
exchange
vesicles between
ratio is >5 so that enzyme per vesicle
Fig. 1. lnterfacial catalysis by PLA, on vesicles in the scooting mode with phase coherence. The enzyme (0) binds irreversibly to the vesicles and hydrolyses only the phospholipid in the outer monolayer of the vesicles. Vesicles that do not contain a bound enzyme are not hydrolysed and the composition of the enzymecontaining vesicles is the same at any time point in the reaction process.
in alternative approach to addressing the ensemble averaging problem is to avoid it all together. For instance, imagine using aggregates that contain a relatively large number of substrates and that the process of enzyme and lipid exchange is completely eliminated. Such a goal has been recently achieved by studying the action of PLA2 in the ‘scooting’ mode. Here, phospholipid vesicles are used which typically contain 10 OOC200 000 substrate molecules. Furthermore, the rate constant for exchange of lipids between the vesicles is usually slow, typically severaf hours. Now if the enzyme remains tightly bound to the surface of the vesicles and if the number of vesicles is in excess over the number of enzymes, the situation shown in Fig. 1 will develop. If each enzyme hydrolyses 100 phospholipids per second, it becomes possible to study the reaction progress in a vesicle containing many thousands of substrates without the need for substrate replenishment. Furthermore, if there is no exchange of components between aggregates, each enzyme-containing vesicle will behave identically in time and all enzymes
837
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Catalysis
and regulation
will be in a common environment at all time points in the reaction progress. Thus, in the scooting mode for a vesicle population of low size-dispersity, the system has a kind of ‘phase coherence’ and the task of ensemble averaging becomes a simple matter of multiplying the amount of product produced in a single enzyme-containing vesicle by the number of such vesicles present in the experiment. In other words, the problem of rapid mixing in substrate aggregates is eliminated by conducting a number of identical ‘closed’ experiments in which each PIAz molecule operates in the isolated world of a single vesicle. That scooting-mode hydrolysis occurs has been extensively demonstrated using the pig pancreatic PL.42 [15,16], and over 30 different PL4$ have recently been shown to operate in the scooting mode [ 171. The requirement for scooting-mode hydrolysis is that the enzyme binds tightly to the vesicle interface. This is the case for vesicles of anionic phospholipids, such as 1,2dimy-ristoyi-glycero-sn-3-phosphomethanol (DMPM). For example, the pig pancreatic PIA, binds to DMPM vesicles some lOlO-fold more tightly than to vesicles of pure zwitterionic phospholipids such as phosphatidylcholine [ 161. Scooting also occurs in phosphatidykholine vesicles that have been doped with small amounts (5-lOmol %> of anionic phospholipids [18-l. The processive nature of the scooting-mode catalysis is a clear indication that the catalytic site of the enzyme is topologically distinct from the interfacial-binding site. If the release of lipolysis products from the catalytic site resulted in the simultaneous desorption of the enzyme from the bilayer, the free enzyme would have no tendency to return to the vesicle to which it was originally bound. Thus, the enzyme must be able to exist in a vesicle-bound state with its cata&tic site unoccupied and ready to accept a new substrate molecule as it processes within the same aggregate.
4 E*+ \
4 S -E*S Phospholipid
-
E*Pz
E*+ \
P
bilayer \
\
E-
l E
Fig. 2. Minimal
kinetic scheme to describe the interfacial catalysis by PLA, according to the Michaelis-Menten formalism. Enzyme in the water layer (El binds to the bilayer to give the vesicle-bound enzyme (E’). The bound enzyme binds a substrate molecule 6) to give the Michaelis complex (E’S), which undergoes chemical transformation to give the enzyme-product complex (E’P). Product dissociation completes the catalytic cycle. At the end of a reaction cycle, E’ can leave the bilayer or remain in the bilayer for additional turnover cycles (processive behavior, shown by the long arrow above the reaction cycle).
Finally, we come to the question of the adequacy of the Michaelis-Menten formalism for the description of inter-
facial catalysis. Based on the above arguments, the simplest scheme that can be constructed to describe the kinetics of interfacial catalysis by PIA contains a step in which the water-soluble enzyme (E) binds to the interface to give the vesicle-bound enzyme (E*; Fig. 2). The bound enzyme can bind a molecule of substrate (S) in its catalytic site to give the Michaelis complex (E’S), which undergoes chemical transformation to give the enzyme-product complex (E*P). Product dissociation completes the catalytic cycle. In this scheme, the catalytic step is considered irreversible because solvent W-exchange studies have shown that the release of the products from E*P is much faster than its conversion to E’S [19]. In the scooting mode, the E to E* step can be ignored and only the interfacial steps (those within the shaded box) need to be considered. In the scooting mode, the enzyme ‘sees’ not the molar concentration of substrate in the experiment, but the surface concentration present in the vesicle to which it is bound. By assuming that the lipid molecules that make up the surface occupy surface areas of similar size, it is possible to represent the surface concentration of substrate by its mole fraction (Xs). According to the classical Michaelis-Menten formalism, the initial enzymatic velocity (i.e. X, = 1 and the mole fraction of products, X,, = 0) is given by: kcat
”
= KM + 1
(1)
where v. is the initial velocity per molecule of enzyme, K, is the interfacial Michaelis constant and bt is the turnover number (where KI and bt have the usual meaning, i.e. when Xs = K, the enzymatic velocity will equal k&2). Thus, equation (1) applies to a single vesicle with a single enzyme molecule irreversibly bound. The total product formed initially per time in the experiment is simply given by v. x CE, where CE is the total amount of enzyme present. In order to describe the reaction progress curve, i.e. the amount of product formed as a function of time, equation (1) can be integrated and the contributions from each enzyme-containing vesicle can simply be summed because each will behave identically in time as long as it contains at most one bound enzyme molecule. Thus:
kit=-In(I-~}+{~-l}{~}
(2)
where Ns is the number of phospholipid molecules in the outer layer of the vesicles (typically 10 000-200000 depending on the vesicle size), and P, and P,, are the amounts of product formed at time t and at the end of the reaction, respectively. Ns& is given by k,..J{Khl(l + 1IKp)) where K, is the equilibrium constant for the dissociation of products from E*P. Equation (2) is analogous to the standard integrated Michaelis-Menten equation, containing a term linear in PJP,, that dominates
Quantitative
and structural
at early reaction times and a term exponential in Pi/P,, that dominates when the mole fraction of substrate becomes limiting. An interesting twist, which is special to vesicle enzymology and is predicted by equation (2), is that the shape of the reaction progress curve will depend on the size of the vesicles (Ns). For example, in large vesicles with one enzyme molecule bound, Xs will remain close to unity for an extended period of time and thus the initial relationship between amount of product released and time will be linear. In contrast, in a small vesicle, still with one bound enzyme molecule, Xs will necessarily change much more rapidly in time and the initial linear relationship may last only a few seconds. In this case, the overall reaction curve takes on a first-order appearance. Such predictions have been experimentally verified [ 20**]. In summary, the standard Michaelis-Menten formalism adequately describes the behavior of PIA undergoing lnterfacial catalysis. The adaption of the formalism to interfacial catalysis requires the following assumptions: at most one enzyme per vesicle; no exchange of any molecules (enzyme, substrate or products) between vesicles; a standard steady-state approximation where the binding steps at the catalytic site are assumed to be faster than the change in surface composition; the intrinsic rate constants shown in Fig. 2 are independent of the surface composition; components are mixed on the surface and are not segregated; and, the surface mole fraction is used to describe the surface concentration. The first two conditions have been experimentally verilied [ 20**] and the others seem reasonable in the light of a lack of anomalous behavior in the reaction progress curves.
Inhibition
of interfacial
catalysis
As discussed in the introduction, inhibitors of PL42 may be useful in the treatment of certain inflammatory disorders in man. In the context of the scheme for interfacial catalysis shown in Fig. 2, a PL$ inhibitor could function in several ways. Irreversible inhibitors could covalently react with the catalytic site so as to block the binding of substrate. pBromophenacyl bromide [21] and SIBLINKS (suicide-inhibitor bifunctionally linked substrates) [22] are examples of such compounds. Agents that covalently modify the interfacial recognition surface of the PIA could function either by preventing the binding of the enzyme to the interface or by causing the PLA2 to bind in a catalytically non-productive manner. Although no examples of the former class of inhibitor have been identilied, an example of the latter class is a synthetic compound called manoalogue [23]. Recent studies have shown that PIA mod&d with manoalogue contains a fully functional catalytic site and is able to bind to vesicles, but the interfacial binding is altered in a way that lowers the turnover of the enzyme in the interface [ 241. For reversible inhibitors, one can envision three classes: (1) an inhibitor present in the interface could promote
analysis
of inhibitors
of phospholipaw
A, Gelb, Berg and lain
the desorption of the bound enzyme (E*+E) - this type of inhibitor might not be specilic for PIA;! as it would not interact directly with the enzyme and would have to be present in the bilayer at a relatively high mole percentage in order to substantially influence the amount of enzyme bound; (2) inhibitors that bind directly to the water-soluble enzyme, E, and prevent its association with the bilayer; and, (3) inhibitors that function in the interface and compete with the substrate for the binding to the active site of E*. Class (2) and (3) inhibitors could be PL$specific and potent as they interact directly with the enzyme. No distinction is made between class (3) inhibitors and a water-soluble compound that binds to the active site of E such that the enzyme-inhibitor complex is still able to associate with the interface because the water-soluble inhibitor would need to function when the enzyme is bound to the interface. If the process of interfacial binding (E+E*) somehow ‘strips’ the inhibitor out of the catalytic site, such a compound could not function to inhibit the interfacial catalysis.
Analysis scooting
of competitive mode
inhibitors
in the
The analysis of reversible PIA inhibitors requires special care because of the various modes of inhibition that are possible. One approach is to study the competitive inhibition that occurs when PIA;! acts on vesicles in the scooting mode [ 25 l *, 261. III this case, the enzyme is irreversibly bound to the vesicle and only those compounds that bind directly in the catalytic site, i.e. class (3) inhibitors, will function as inhibitors. Class (2) inhibitors may also work in this system if they can compete with the vesicles for binding to E. These points are important in light of the numerous inhibitors of PIA;! that have been reported in the literature which have more recently been shown to function as non-specilic modulators of lnterfacial catalysis [25”, 26, 271. For example, many lipophilic agents till function as inhibitors of PIA* when this is acting on vesicles of zwltterionic substrate. In this situation, the enzyme is weakly bound and the fraction of enzyme bound is sensitive to the presence of lipophilic additives. Characteristic features of these class (1) inhibitors are that they work only when the enzyme is partitioned between the E and E* forms, a high mole percentage of compound is needed, and they fail to inhibit the catalysis by PIA;! in the scooting mode. In fact, one of the advantages of the scooting-mode analysis is that not only is it carried out in the absence of additives, but the enzyme kinetics are insensitive to the presence of lipophillc substances that do not bind to the enzyme, even when such compounds are present in the vesicle at concentrations up to roughly 20mol%. Examples of non-speciIic modulators of interfacial catalysis include alkanols, fatty acids, local anesthetics, butyrophenones, mepacrine, aristolochic acid and quinacrine [ 25.0, 271. It is unlikely that class (1) inhibitors could be useful as drugs in vivo as they would have to be present in the bilayer at rel-
839
840
Catalysis
and regulation
atively high concentrations and would therefore be expected to produce a number of non-specific effects. The equations that describe competitive inhibition by class (3) inhibitors in the scooting mode are particularly simple. Different equations are used depending on the size of the vesicles.
Equation (3) is appropriate for large vesicles and gives the ratio of initial enzymatic velocities in the absence (g) and presence (v’s) of an inhibitor as a function of the interfacial equilibrium constant for the dissociation of the inhibitor from E*I (Kt), the interfacial KM, and the mole fraction of inhibitor (XI>. As discussed above, in a large vesicle, the initial velocity can be measured and the inhibitor is in competition with the substrate because the concentration of product is near zero. For this reason, the degree of inhibition depends on the value of both Kt and KIMas shown in equation (3). Equation (4) is appropriate for small vesicles, in which the initial velocity is not easily observed and the concentration of products is sig niiicant. In this situation, the inhibitor must compete with the product and the degree of inhibition depends on the values of Kr and Kp. These equations have been experimentally veriiied for a number of competitive inhibitors of PIA;! [25”,26].
A direct method for the analysis of ligand binding to phospholipase A, in the interface
A second method that has been used to characterize the binding of a ligand to the active site of PIAz in the interface is to determine whether or not the ligand protects the catalytic site of the enzyme from attack by an alkylating agent [25**]. In this approach, the enzyme is fully bound to an interface made up of a neutral surface diluent. Such a compound is delined as an amphiphile that forms an interface to which PIA;! can bind but does not bind well to the catalytic site on the enzyme. A number of compounds were examined for their ability to function as a neutral diluent and one compound, 2-hexadecyl-glycero-sn-3-phosphocholine (2H-GPC) was found to have the following properties. 2H-GPC formed micelles to which pig pancreatic PIA;! bound as demonstrated by fluorescence studies [25**]. In the prescence of a st&ently high concentration of 2H-GPC (typically 3mM), all of the PI& was bound to the interface. Next, the rate of alkylation of the active-site his&line residue (His48) by well known PIA inactivators, such as pni-
trophenacyl bromide, was studied. The rate of enzyme alkylation was found to be the same for the PIA;! either in aqueous solution or fully bound to 2H-GPC rnicelles. This result indicates that 2H-GPC does not occupy the catalytic site on E*, and that the binding of the akybing agent to the catalytic site and the binding of PI-42 to the interface are not synergistic [ 25”]. Thus, 2H-GPC has the properties of a neutral diluent and micelles of this compound provide a useful matrix with which to study the binding of other ligands to the catalytic site of E*. When the mole fraction of a ligand in the 2H-GPC interface is equal to the dissociation constant (in units of mole fraction) for the E*L complex (L, l&and), half the enzyme is present as E* and half as E*L Under these conditions, the half-time for alkylation in the presence of the ligand will be twice that measured in the absence of the ligand. This method of measuring lig and dissociation constants for PIA has been previously applied to the water-soluble form of the enzyme [211 and the extension to the study of E* as described here presents no major problems. Using this approach, the dissociation constants for a variety of ligand have been measured [25”]. For example, the K, for a potent enantiomer of a phosphate-containing transition-state analog was found to have a value of 0.001. This enantiomer, which has a glycerol backbone stereochemistry that is similar to that found in naturally occuring phospholipids (m-3) is a much better inhibitor than its enantiomer (sn1). Using this value of the Kt, a value of KM = 0.3 mole fraction for the DMPM substrate could be obtained from the inhibition data and equation (3). The KP for the combination of lipolysis products (fatty acid and lysophospholipid) was directly measured to be 0.025 using the protection method. In good agreement with this, a value of KP = 0.036 was obtained from the inhibition data with the transition-state analog in small vesicles using equation (4) together with the K, value of 0.001. Because the DMPM substrate undergoes catalytic tmnsformation, it is not possible to evaluate its interaction with E* using the protection method. However, in addition to dete rmining the Khl value from the inhibition data (see above), the value of this Michaelis constant could be obtained by applying the scooting-mode analysis to DMPM vesicles in the presence of the neutral diluent 2H-GPC was added to DMPM vesicles up to a mole fraction of -0.3 without solubilizing the vesicles. This provided a way of lowering the surface concentration (or mole fraction) of substrate that the bound enzyme ‘sees’. The resulting decrease in the initial enzymatic velocity that we observed could only have arisen from surface dilution because 2H-GPC does not function as a competitive inhibitor nor does it disrupt the vesicles when present up to a mole fraction of about 0.3. This analysis yielded a value of KM = 0.3 mole fraction [25**]. This number agrees well with the value of KM determined from the studies with several competitive inhibitors. It is interesting to note that although the PIA is embedded in a maximally packed array of DMPM substrate in the vesicle, the Khl for the substrate is not much lower than the substrate concentration (initially 1.0 mole fraction),
Quantitative
and structural
analysis
and the presence of trace amounts of potent inhibitors can lead to significant inhibition of the interfacial catalysis. This statement has obvious consequences for the design of medicinally important PLA2 inhibitors.
Structural studies of phospholipase inhibitor complexes
A,-
By using inhibitors of PLA2 with short alkyl chains, it has been possible to obtain crystals of three different enzyme-inhibitor complexes. Thunnissen et al. [28**] determined the crystal structure of the pig pancreatic PLk* in a complex with an inhibitor in which the enzyme-susceptible ester was replaced with an amide. The honey bee and Chinese cobra (Nuju nuju utru) venom PLA*s have been crystallized in the presence of a phosphonate-containing phospholipid analogue [29*=-31**]. The active-site region of the cobra venom structure is shown in Fig. 3. It can be seen that one of the inhibitor’s non-bridging phosphonate oxygens is bound to the Ca*+ ion and the other oxygen atom is hydrogenbonded to the active-site histidine residue. This result is consistent with the previously proposed mechanism for PIA2 catalysis [1,21] in which a histidine-bound water molecule attacks the carbonyf group of the substrate’s m2 ester to form a tetrahedral intermediate with the oxyanion of this intermediate bound to the Ca2+ ion. In addition, the amide NH of Gly30 donates a hydrogen bond to the oxyanion. The fact that the pro-S oxygen atom of the
of inhibitors
of phospholipase
A, Celb,
Berg
and
lain
inhibitor’s phosphate group is bound to the Ca2+ ion (Fig. 3) suggests that the cofactor helps to anchor the substrate in a catalytically productive manner in addition to its role in electrophilic catalysis. A tyrosine residue donates a hydrogen bond to the pro-R oxygen of the phosphate group in the cobra venom enzyme, whereas in the bee venom enzyme this function is fulhlled by a threonine residue. A detailed view of the Ca2+ ligation cage in the presence and absence of the inhibitor is shown in Fig. 4. In the uninhibited enzyme (Fig. 4a), the Ca2+ ion is heptacoordinate, with live ligands coming from the protein and two from solvent waters. In addition, a third water molecule is seen bound to the histidine residue. When the enzyme is bound to the phosphonate inhibitor, these three water molecules are replaced with inhibitor oxygen atoms with no detectable deformation of the protein structure (Fig. 4b). It is interesting to note that the constellation of active-site residues and the conformation of the bound inhibitor are virtually superimposable for the cobra- and bee-venom structures, despite the fact that the global structure of the bee-venom enzyme differs dramatically from the common structural architecture seen with other extracellular PIA2s. If it is imagined that the ester substrate binds to the enzyme with its carbonyf group liganded to the Ca2+ ion and with an attacking water molecule hydrogen bonded to the histidine, the ester would be required to rotate out of the plane defined by the trigonal carbonyl and the bridging oxygen. This can readily be seen from Fig. 3 by overlaying the ester onto the bound conforma-
.. & i
-
Fig. 3. Stereo-diagram of cobra venom metal ligations are indicated by dotted onto the phosphonate by least-squares enzyme structure.
phospholipase A, with a bound lines. Also shown is the structure superpositioning of the entire
phosphonate phospholipid analogue. The hydrogen bonds and of the bound amide inhibitor [27**1. The amide was overlayed pig pancreatic enzyme structure onto the entire cobra venom
841
842
Catalysis
and regulation
\
,
phospholipase A, in the absence of phosphonate inhibitor. The protein Fig. 4. (a) Coordination structure of the Ca 2+ ion in cobra venom donates five ligands to the metal coming from the amide carbonyl groups of residues 28, 30 and 32, as well as bidentate coordination from the side chain of Asp49. The three water molecules are denoted as W. (b) Coordination structure of the Ca2+ ion in the presence of the bound phosphonate phospholipid analogue. P2 denotes the phosphonate group and P3 denotes the phosphate group. The hydrogen bond between the oxygen of P3 and a tyrosine is not shown.
tion of the phosphonate. Such a rotation may be possible with the ester but would be prohibitively large for the amide. Indeed, the crystal structure of the enzymebound amide shows that the inhibitor is tilted in the active site relative to the phosphonate [28**]. This can be seen in Fig. 3 where the amide structure has been overlaid onto the bound phosphonate. The amide carbonyl group is bound to the Ca*+ ion and the amide NH is hydrogen-bonded to histidine. This later interaction leads to the displacement of the water molecule that is hydrogen-bonded to the histidine in the uninhibited enzyme. This hydrogen bond may also explain why amidecontaining phospholipid analogues bind 100-1000 times more tightly to the enzyme than the corresponding esters [32-l. Based on the fact that the structures of the enzymes in the presence and absence of the bound inhibitor are virtually superimposable on each other [ 29**], it is tempting to propose that a sign&ant conformation change does not occur in the enzyme upon binding to the lipid interface. It is clear from these crystal structures that a phospholipid molecule must diffuse from the plane of the membrane bilayer through a hydrophobic channel in the enzyme that contains the catalytic machinery. It is possible that there is a tight seal formed between the substrate interface and the Wet-facial recognition surface of the enzyme. It has been shown that the binding of the enzyme to the interface occurs with desolvation of the protein and lipid surfaces that contact each other [33]. This desolvation may allow the phospholipid to be transferred down the
hydrophobic channel with minimal contact with bulk solvent.
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KIM DK, SUH PG, Rnr SH: PuriIicatIon and Some Roperties of a PhosphoIipase Aa from Bovine Platelets. Bibcbem Biophys Res Commun 1991, 174:189-l%.
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27:392>3931. 14.
J, DE m GH: Kinetics of Binding of Phosphotipase Aa to Lipid/Water Interfaces and its ReIationship to IntertkiaI Activation. Biocbim Biq&ys Acta 1988,
MK, BERG 0: The Kinetics of Interfacial Catalysis by Phospholipase Aa and Regulation of Interfacial Activation: Hopping Versus Scooting. Biocbim Biophvs Actu 1989,
JAIN
1002:127-156. 16.
J.UN MK,
Ro~~t-6
J, JAHAGIRDAR
DV,
MARECEK JF,
RAMIREZ
F: Kinetics of Interfacial Catalysis by Phospholipase Aa in Intravesicle Scooting Mode, and Heterofusion of Anionic and Zwitterionic Vesicles. Biochim Biophvs Acti 1986, 86Oz435-447. 17.
18. ..
HM: Interfacial Catalysis by Phospholipase Aa: Monomeric Enzyme is Fully CataIyticaIIy Active at the BiIayer Interface. Bitt cbf?mer?y 1991, 30:7330-7340.
JUN
MK.
RANAD~VE
G,
Yu
B-Z,
VERHEIJ
GHOMASHCH~ F, Yu B-Z, BERG 0, JAIN MK, GELB MH: Interfacial CataIysii by Phospholipase A,: Substrate Specikity in Vesicles. Biochemistry 1991, u):7318-7329.
This paper discusses the substrate specilicity of PIA, on vesicles in the scooting mode, which provides the only reliable means of obtaining the specificity constants (k&Ku). A useful review of previous work on substrate-specificity studies with PIAz is also included. 19.
20. ..
GHOMASHCHI
F, O’HARE
T, CLARY D, GELB
MH:
WASHBURNWH, DENNIS EA: Suicide-inhibitor BifunctionaBy Linked Substrates (SIBLINKS) as Phospholipase Aa Inhibitors. / Biol Cbem 1991, 266:5042-5048. REYNOIDSLJ, MORGAN BP, Hm Gq M~HEUCH ED, DE= W: PhosphoIipase 4 Inhibition and ModRication by Monoalogue. J Am Ckm SCX 1988, 110:5172-5177. GHOMASHCHI F, Yu B-Z, JAIN MK, GEIB MH: Kinetic Characterization of PhosphoIipase A2 ModIEed by Manoalogue. 1991,
in press.
GN, BERG 0: InterfaciaI Catalysis by PhosphoIipase A2: Dissociation Constants for Calcium, Substrate, and Competitive Inhibitors. Biochemistry 1991, 30:7306-7317.
JAIN MK, Yu B-Z, R~GER~ J, fw.uxv~
Interfacial
J.w MK, YUAN W, GELB MH: Competitive Inhibition of Pbospholipase A2 in Vesicles. Bfbcbemm 1989, 28:4135-4139. JALN MK, JAHAGUU%R DV: Action of Phospholipase A2 on BiIayers. ERect of Inhibitors. Biocbim Bi@ys Acfa 1985, 814:31+326.
28. ..
THUNNISSEN MMGM, A6 E, KU KH, DRENIH J, DIG KULPERS OP, DIJK~~AN R, DE m GH, VERHEIJ HM:
phospholipid bound as to why the amide 29. ..
Scorr PB:
DI
to an enzyme. phospholipid
The structure provides analogues bind tightly
WH~?E SP, Owt~ou%~
Z, YUAN W, GELB MH, SIGNER
Aa. Science 1990, 250:1541-1546. This series of papers [28**-30**] describes the first crystal structures of a phospholipid bound to an enzyme. The strucNm provide strong evidence for a catalytic mechanism for the esterolysis. 30. ..
SCOTTDL, O~INOWSKI Z, GELB MH, SIGNER PB: Crystal Struchue of Bee-venom PhosphoIipase A2 In a Complex with a Transition-state AnaIogue. Science 1990, 250:15631566.
This series of papers [28**-30**] describes the first crystal strucNres of a phospholipid bound to an enzyme. The structures provide strong evidence for a catalytic mechanism for the esteroh&. 31. ..
WHITT
SP, Scorr
DI
C&wt~owsto
Z, GELE MH,
This series of papers [28**-30**] describes the Iirst ctystaJ StrucNre of a phospholipid bound to an enzyme. The structures provide strong evidence for a catalytic mechanism for the esterolysis. RANSAC s, RMERE c, HAAS GH: Competitive
Soutt~ JM, G.wcm C, VERGER R, DE btbibition of Lipolytic Enzymes. 1. A
This paper discusses the theory for competitive inhibition mixed micelIes. The other papers that follow in the series amples of inhibitors and their analysis in mixed micelles.
JJ,
PIETERSON
WA,
DE
the Active Site of PhosphoIipase E&1446-1454.
HAAS
GH:
Histidine
AZ Biabemishy
at 1974,
SIGIER PB:
CyrstaI Structure of Cobra-venom Phospholipase A2 in a Complex with a Transition-state AnaIogue. Science 1990, 250:1560-1563.
BERGOG, Yu B-Z, ROGERS Jq JAIN MK: Interfacial Catalysis by Phospholipase Aa: Determination of the Interfacial Kinetic Rate Constants. Biochemistry 1991, 30:72837297.
VOLWERK
some clues to PL42.
Interfacial CataIysis: the Mechanism of Phospholipase
32. ..
accomplished for an enzyme that operates in an interface.
BW, X-ray
Structure of Phospholipase A2 Complexed with a Substratederived Inhibitor. Nuhtre 1990, 347689691. This paper and [29**,30**] describe the first crystal structures of a
Catalysis by Phospholipase Aa: Evaluation of the Interfacial Rate Constants by Steady-State Isotope Effect Studies. Bichzbemisf?y 1991, 30:7298-7305.
This paper develops the basic concept of scooting kinetics for interfacial catalysis on vesicles. Both the experimentaJ verification of scooting and the theoretical description of the process are detailed. Data from this paper and from papers that follow it in the series are puked together in order to extract most of the interfacial kinetic rate constants for the steps shown in Fig. 2. This is the first time that this has been 21.
27.
JAM MK. ROGER
%0:5162. 15.
A2 Gelb, Berg and Jain
This paper describes the theory and experimental observations of competitive inhibition in the scooting mode on both large and small vesicles. Also developed is the concept of a neutral diktent and the use of such a compound to measure the dissociation constants for a variety of ligands that interact with PL42 in the interface.
DC, NICHOLS JW: Characterization of PhosphoIipid Transfer between Mixed Phospholipid-Bile Salt Miceks. Biochem~ 1990, 29:87-. DA, SHOEMAKER
This paper is the second in a series that reports the first measurements of the rates of phospholipid exchange in mixed micelles. 13.
..
2595734-5739.
FUUNGTON
of phospholipase
Biocbemishy 25.
11.
of inhibitors
Kinetic Model Applicable to Water-insoluble Competitive Inhibitors. Biochim Bic#&ys Acta 1990, 10435746.
33.
of PIA in provide ex-
Dehydration of the Lipid-Protein Microinterface on Binding of Phospholipase A2 to Lipid BIIayers.
J,UN MK, VAZ WIG: Biocbim
Bi@ys
Acta
1987, 905:1-8.
MH Gelb. Department of Chemistry, BG-10, University of Washmgton, Seattle, WA 98195, USA 0 Berg, Department of Molecular Biology, Uppsakt University, Sweden. hlK Jain, Department of Chemistrv and Biochemistry, University of Delaware 19716, USA
643
and structural analysis of inhibitors ‘phospholipase A2 H. Gelb, Otto
Berg and Mahendra
of Washington, Seattle, Washington, and University of Delaware,
of
K. Jain
USA, Uppsala University, Newark, Delaware, USA
Uppsala,
Sweden
The evaluation of competitive inhibitors of phospholipase A, is best carried out by analysing the enzyme under conditions in which it binds irreversibly to the substrate vesicle and catalyses a processive reaction in which all of the phospholipid molecules on the outer layer of the vesicle are hydrolysed. Recent structural analyses of phospholipase AZ-inhibitor complexes suggest a mechanism for the catalytic site.
Current
Opinion
in Structural
Biology
1:836-843
zymes are thought to play a role in degenerate joint diseases. In addition, PLA2 inhibitors may block the liberation of arachidonic acid during the eicosanoid cascade. The scope of this review is aimed at recent developments in the kinetic and structural analysis of PLA2.
Introduction
Extracellular secretory phospholipase AZS (P&s) are small (14kD), water-soluble enzymes that hydrolyse the ester at the ~2-2 position of phospholipids. Abundant forms are the pancreatic P&s, which are involved in lipid digestion, and the enzymes found in a variety of animal venoms, which contribute to the toxicity of these substances. The properties of these enzymes have been reviewed [l-3]. More recently, PLA2s have been detected in a variety of different animal fluids and tissues and have been shown to be secreted from certain cells such as platelets. The role of these enzymes in inflammation and signal transduction is currently under intensive study [4]. All of these extracellular PLA2s require approximately 1O-4 M calcium for activity and contain multiple disulfide bonds.
Does phospholipase A, obey the Michaelis-Menten formalism?
The answer to this question is yes. This is reassuring because the Michaelis-Menten formalism has been useful in describing the quantitative behavior of essentially every known enzyme that operates in a homogeneous environment. Thus, rather than abandoning tradition, a serious effort should be made to use this basic paradigm of enzyme kinetics for the kinetic analysis of inter-facial catalysis by PIA*. The crux of the problem in achieving this is that the substrate is present in aggregates, dispersed as micelles or vesicles, which have a finite size. The kinetic analysis of any system requires a perfect mixing of all reactants so that every enzyme on average ‘sees’ the same environment of substrate and product at any particular time. In solution enzymology, rapid mixing is normally not a concern. When studying interfacial catalysis, however, special care has to be taken to ensure that all enzymes operate in a common environment.
A novel PI.& with a Mr -88 000 has been recently detected in the cytoplasm of macrophages [5-8], kidney [9] and platelets [lo]. In the presence of submicromolar amounts of calcium, this enzyme becomes catalytically active and is tmnslocated from the cytoplasm to the membrane of cells. This enzyme may serve to release arachidonic acid from the membrane pool for the biosynthesis of the eicosanoids. Because naturally occurring phospholipids are always present in interfaces, PLA$ have evolved to be considerably more active on substrate aggregates. The quantitative analysis of the interfacial catalysis by PLA2 is more challenging than that of enzyme systems that operate in a homogeneous aqueous environment. The inherent heterogeneity of the lipid-water interface often produces anomalous kinetic behavior which in turn has led to misconceptions about substrate preferences and inhibition with this class of enzymes. Inhibitors of interfacial catalysis by PIA;! may be medicinally significant as these en-
For example, consider the action of PI.& on a mixedmicellar substrate, a commonly studied system, in which the phospholipid molecules are dispersed in an ensemble of detergent micelles [ 1,2,11]. If there is no exchange of enzyme or lipid between these micelles, the reaction would quickly come to a halt after the 10 or so substrate molecules in a mixed micelle containing the enzyme have been hydrolysed. As a typical turnover number for a PIA2
Abbreviations 2H-GPC-2-hexadecyl-glycero-sn-3-phosphocholine;
DMPM-1,2-dimyristoyl-glycero-sn-3-phosphomethanol; 836
1991,
@
Current
Biology
Ltd
ISSN
0959440X
PL4*-phospholipase
A,.
Quantitative
and structural
analysis
of inhibitors
A, Celb, Berg and lain
of phospholipase
is well over lCOs- *, the contents of a single mixed-micelle containing PM, will be signiticantly altered in just a few milliseconds. If the lipolysis products are removed from the micelle to other miceUes by lipid exchange and the substrate is replenished by exchange from miceUes with no enzymes, the reaction can continue for an extended period. Only if these exchange processes are fast on the millisecond timescale will the observed steadystate enzymatic velocity be free of distortions resulting from low miceUar exchange rates, and will all of the micelles change in composition in a uniform way, with all enzyme molecules present in a common environment at any point in time. Alternatively, the enzyme could hop among the ensemble of miceUes. For there to be no distortion in enzyme velocity in this situation, the enzyme must leave the miceUe before it has depleted the substrate, i.e. the rate constant for the desorption of the PIA* from the miceUe must be larger than the rate constant for change in the composition of a single miceUe. Only in this fast hopping mode will all of the miceUes change in composition in a uniform way and will all enzyme molecules exist in a common environment. These requirements of either fast lipid or fast enzyme exchange are hard to verify experimentaUy. The only reported rates of phospholipid exchange in mixed miceUes come from recent studies by Nichols and coworkers [12**,13]. Here, the exchange rates of fluoresce&y labeled phospholipids in miceUes of bile salts were measured by a fluorescence dequenching approach. For long-chain phospholipids, which essentially never enter the aqueous phase, intermiceuar exchange occurs by a process that requires the collision of miceUes. With the concentrations of components that are typically used in PLA, experiments (510 mM detergent, l-2 mM phospholipids), the half-times for exchange range from m 100 ms to several seconds, depending on the structure of the bile salt. This is clearly not fast on the l-5ms timescale required for rapid averaging during PIAz turnover. The problem may be even more complicated as one reaUy needs to measure the rate of lipid exchange not in a pure mixed-miceUe but in one that contains a bound enzyme. One could imagine that a bound PLA2 might ‘trap’ lipid molecules that make up a protein-lipid complex. The rate constant for PIAz desorption from miceUes and vesicles has been measured by stopped-Uow fluorescence [ 141. An upper-limit value of about 4s-t was measured and found to be remarkably insensitive to the nature of the phospholipid interface. Thus, it appears that the desorption of enzyme from a miceUe may not be fast on the few millisecond timescale. In summary, one cannot safely conclude that exchange of components in miceUar enzymology will in all cases be fast enough to ensure that the intrinsic catalytic properties of the PLA2 are being measured. Rates of enzymatic turnover in miceUes may be more a reflection of the rates of exchange of lipid or enzyme among the ensemble of substrate aggregates. It is the problem of substrate aggregates of Unite size and the requirement for rapid mixing, that can lead to problems in the kinetic analysis of interfacial catalysis.
0
Experimentally 1. Enzyme 2. Substrate vesicles.
verified does
not
and
3. The vesicle:enzyme there is at most
constraints: exchange
products
one
do
(‘hop’) not
between
exchange
vesicles between
ratio is >5 so that enzyme per vesicle
Fig. 1. lnterfacial catalysis by PLA, on vesicles in the scooting mode with phase coherence. The enzyme (0) binds irreversibly to the vesicles and hydrolyses only the phospholipid in the outer monolayer of the vesicles. Vesicles that do not contain a bound enzyme are not hydrolysed and the composition of the enzymecontaining vesicles is the same at any time point in the reaction process.
in alternative approach to addressing the ensemble averaging problem is to avoid it all together. For instance, imagine using aggregates that contain a relatively large number of substrates and that the process of enzyme and lipid exchange is completely eliminated. Such a goal has been recently achieved by studying the action of PLA2 in the ‘scooting’ mode. Here, phospholipid vesicles are used which typically contain 10 OOC200 000 substrate molecules. Furthermore, the rate constant for exchange of lipids between the vesicles is usually slow, typically severaf hours. Now if the enzyme remains tightly bound to the surface of the vesicles and if the number of vesicles is in excess over the number of enzymes, the situation shown in Fig. 1 will develop. If each enzyme hydrolyses 100 phospholipids per second, it becomes possible to study the reaction progress in a vesicle containing many thousands of substrates without the need for substrate replenishment. Furthermore, if there is no exchange of components between aggregates, each enzyme-containing vesicle will behave identically in time and all enzymes
837
838
Catalysis
and regulation
will be in a common environment at all time points in the reaction progress. Thus, in the scooting mode for a vesicle population of low size-dispersity, the system has a kind of ‘phase coherence’ and the task of ensemble averaging becomes a simple matter of multiplying the amount of product produced in a single enzyme-containing vesicle by the number of such vesicles present in the experiment. In other words, the problem of rapid mixing in substrate aggregates is eliminated by conducting a number of identical ‘closed’ experiments in which each PIAz molecule operates in the isolated world of a single vesicle. That scooting-mode hydrolysis occurs has been extensively demonstrated using the pig pancreatic PL.42 [15,16], and over 30 different PL4$ have recently been shown to operate in the scooting mode [ 171. The requirement for scooting-mode hydrolysis is that the enzyme binds tightly to the vesicle interface. This is the case for vesicles of anionic phospholipids, such as 1,2dimy-ristoyi-glycero-sn-3-phosphomethanol (DMPM). For example, the pig pancreatic PIA, binds to DMPM vesicles some lOlO-fold more tightly than to vesicles of pure zwitterionic phospholipids such as phosphatidylcholine [ 161. Scooting also occurs in phosphatidykholine vesicles that have been doped with small amounts (5-lOmol %> of anionic phospholipids [18-l. The processive nature of the scooting-mode catalysis is a clear indication that the catalytic site of the enzyme is topologically distinct from the interfacial-binding site. If the release of lipolysis products from the catalytic site resulted in the simultaneous desorption of the enzyme from the bilayer, the free enzyme would have no tendency to return to the vesicle to which it was originally bound. Thus, the enzyme must be able to exist in a vesicle-bound state with its cata&tic site unoccupied and ready to accept a new substrate molecule as it processes within the same aggregate.
4 E*+ \
4 S -E*S Phospholipid
-
E*Pz
E*+ \
P
bilayer \
\
E-
l E
Fig. 2. Minimal
kinetic scheme to describe the interfacial catalysis by PLA, according to the Michaelis-Menten formalism. Enzyme in the water layer (El binds to the bilayer to give the vesicle-bound enzyme (E’). The bound enzyme binds a substrate molecule 6) to give the Michaelis complex (E’S), which undergoes chemical transformation to give the enzyme-product complex (E’P). Product dissociation completes the catalytic cycle. At the end of a reaction cycle, E’ can leave the bilayer or remain in the bilayer for additional turnover cycles (processive behavior, shown by the long arrow above the reaction cycle).
Finally, we come to the question of the adequacy of the Michaelis-Menten formalism for the description of inter-
facial catalysis. Based on the above arguments, the simplest scheme that can be constructed to describe the kinetics of interfacial catalysis by PIA contains a step in which the water-soluble enzyme (E) binds to the interface to give the vesicle-bound enzyme (E*; Fig. 2). The bound enzyme can bind a molecule of substrate (S) in its catalytic site to give the Michaelis complex (E’S), which undergoes chemical transformation to give the enzyme-product complex (E*P). Product dissociation completes the catalytic cycle. In this scheme, the catalytic step is considered irreversible because solvent W-exchange studies have shown that the release of the products from E*P is much faster than its conversion to E’S [19]. In the scooting mode, the E to E* step can be ignored and only the interfacial steps (those within the shaded box) need to be considered. In the scooting mode, the enzyme ‘sees’ not the molar concentration of substrate in the experiment, but the surface concentration present in the vesicle to which it is bound. By assuming that the lipid molecules that make up the surface occupy surface areas of similar size, it is possible to represent the surface concentration of substrate by its mole fraction (Xs). According to the classical Michaelis-Menten formalism, the initial enzymatic velocity (i.e. X, = 1 and the mole fraction of products, X,, = 0) is given by: kcat
”
= KM + 1
(1)
where v. is the initial velocity per molecule of enzyme, K, is the interfacial Michaelis constant and bt is the turnover number (where KI and bt have the usual meaning, i.e. when Xs = K, the enzymatic velocity will equal k&2). Thus, equation (1) applies to a single vesicle with a single enzyme molecule irreversibly bound. The total product formed initially per time in the experiment is simply given by v. x CE, where CE is the total amount of enzyme present. In order to describe the reaction progress curve, i.e. the amount of product formed as a function of time, equation (1) can be integrated and the contributions from each enzyme-containing vesicle can simply be summed because each will behave identically in time as long as it contains at most one bound enzyme molecule. Thus:
kit=-In(I-~}+{~-l}{~}
(2)
where Ns is the number of phospholipid molecules in the outer layer of the vesicles (typically 10 000-200000 depending on the vesicle size), and P, and P,, are the amounts of product formed at time t and at the end of the reaction, respectively. Ns& is given by k,..J{Khl(l + 1IKp)) where K, is the equilibrium constant for the dissociation of products from E*P. Equation (2) is analogous to the standard integrated Michaelis-Menten equation, containing a term linear in PJP,, that dominates
Quantitative
and structural
at early reaction times and a term exponential in Pi/P,, that dominates when the mole fraction of substrate becomes limiting. An interesting twist, which is special to vesicle enzymology and is predicted by equation (2), is that the shape of the reaction progress curve will depend on the size of the vesicles (Ns). For example, in large vesicles with one enzyme molecule bound, Xs will remain close to unity for an extended period of time and thus the initial relationship between amount of product released and time will be linear. In contrast, in a small vesicle, still with one bound enzyme molecule, Xs will necessarily change much more rapidly in time and the initial linear relationship may last only a few seconds. In this case, the overall reaction curve takes on a first-order appearance. Such predictions have been experimentally verified [ 20**]. In summary, the standard Michaelis-Menten formalism adequately describes the behavior of PIA undergoing lnterfacial catalysis. The adaption of the formalism to interfacial catalysis requires the following assumptions: at most one enzyme per vesicle; no exchange of any molecules (enzyme, substrate or products) between vesicles; a standard steady-state approximation where the binding steps at the catalytic site are assumed to be faster than the change in surface composition; the intrinsic rate constants shown in Fig. 2 are independent of the surface composition; components are mixed on the surface and are not segregated; and, the surface mole fraction is used to describe the surface concentration. The first two conditions have been experimentally verilied [ 20**] and the others seem reasonable in the light of a lack of anomalous behavior in the reaction progress curves.
Inhibition
of interfacial
catalysis
As discussed in the introduction, inhibitors of PL42 may be useful in the treatment of certain inflammatory disorders in man. In the context of the scheme for interfacial catalysis shown in Fig. 2, a PL$ inhibitor could function in several ways. Irreversible inhibitors could covalently react with the catalytic site so as to block the binding of substrate. pBromophenacyl bromide [21] and SIBLINKS (suicide-inhibitor bifunctionally linked substrates) [22] are examples of such compounds. Agents that covalently modify the interfacial recognition surface of the PIA could function either by preventing the binding of the enzyme to the interface or by causing the PLA2 to bind in a catalytically non-productive manner. Although no examples of the former class of inhibitor have been identilied, an example of the latter class is a synthetic compound called manoalogue [23]. Recent studies have shown that PIA mod&d with manoalogue contains a fully functional catalytic site and is able to bind to vesicles, but the interfacial binding is altered in a way that lowers the turnover of the enzyme in the interface [ 241. For reversible inhibitors, one can envision three classes: (1) an inhibitor present in the interface could promote
analysis
of inhibitors
of phospholipaw
A, Gelb, Berg and lain
the desorption of the bound enzyme (E*+E) - this type of inhibitor might not be specilic for PIA;! as it would not interact directly with the enzyme and would have to be present in the bilayer at a relatively high mole percentage in order to substantially influence the amount of enzyme bound; (2) inhibitors that bind directly to the water-soluble enzyme, E, and prevent its association with the bilayer; and, (3) inhibitors that function in the interface and compete with the substrate for the binding to the active site of E*. Class (2) and (3) inhibitors could be PL$specific and potent as they interact directly with the enzyme. No distinction is made between class (3) inhibitors and a water-soluble compound that binds to the active site of E such that the enzyme-inhibitor complex is still able to associate with the interface because the water-soluble inhibitor would need to function when the enzyme is bound to the interface. If the process of interfacial binding (E+E*) somehow ‘strips’ the inhibitor out of the catalytic site, such a compound could not function to inhibit the interfacial catalysis.
Analysis scooting
of competitive mode
inhibitors
in the
The analysis of reversible PIA inhibitors requires special care because of the various modes of inhibition that are possible. One approach is to study the competitive inhibition that occurs when PIA;! acts on vesicles in the scooting mode [ 25 l *, 261. III this case, the enzyme is irreversibly bound to the vesicle and only those compounds that bind directly in the catalytic site, i.e. class (3) inhibitors, will function as inhibitors. Class (2) inhibitors may also work in this system if they can compete with the vesicles for binding to E. These points are important in light of the numerous inhibitors of PIA;! that have been reported in the literature which have more recently been shown to function as non-specilic modulators of lnterfacial catalysis [25”, 26, 271. For example, many lipophilic agents till function as inhibitors of PIA* when this is acting on vesicles of zwltterionic substrate. In this situation, the enzyme is weakly bound and the fraction of enzyme bound is sensitive to the presence of lipophilic additives. Characteristic features of these class (1) inhibitors are that they work only when the enzyme is partitioned between the E and E* forms, a high mole percentage of compound is needed, and they fail to inhibit the catalysis by PIA;! in the scooting mode. In fact, one of the advantages of the scooting-mode analysis is that not only is it carried out in the absence of additives, but the enzyme kinetics are insensitive to the presence of lipophillc substances that do not bind to the enzyme, even when such compounds are present in the vesicle at concentrations up to roughly 20mol%. Examples of non-speciIic modulators of interfacial catalysis include alkanols, fatty acids, local anesthetics, butyrophenones, mepacrine, aristolochic acid and quinacrine [ 25.0, 271. It is unlikely that class (1) inhibitors could be useful as drugs in vivo as they would have to be present in the bilayer at rel-
839
840
Catalysis
and regulation
atively high concentrations and would therefore be expected to produce a number of non-specific effects. The equations that describe competitive inhibition by class (3) inhibitors in the scooting mode are particularly simple. Different equations are used depending on the size of the vesicles.
Equation (3) is appropriate for large vesicles and gives the ratio of initial enzymatic velocities in the absence (g) and presence (v’s) of an inhibitor as a function of the interfacial equilibrium constant for the dissociation of the inhibitor from E*I (Kt), the interfacial KM, and the mole fraction of inhibitor (XI>. As discussed above, in a large vesicle, the initial velocity can be measured and the inhibitor is in competition with the substrate because the concentration of product is near zero. For this reason, the degree of inhibition depends on the value of both Kt and KIMas shown in equation (3). Equation (4) is appropriate for small vesicles, in which the initial velocity is not easily observed and the concentration of products is sig niiicant. In this situation, the inhibitor must compete with the product and the degree of inhibition depends on the values of Kr and Kp. These equations have been experimentally veriiied for a number of competitive inhibitors of PIA;! [25”,26].
A direct method for the analysis of ligand binding to phospholipase A, in the interface
A second method that has been used to characterize the binding of a ligand to the active site of PIAz in the interface is to determine whether or not the ligand protects the catalytic site of the enzyme from attack by an alkylating agent [25**]. In this approach, the enzyme is fully bound to an interface made up of a neutral surface diluent. Such a compound is delined as an amphiphile that forms an interface to which PIA;! can bind but does not bind well to the catalytic site on the enzyme. A number of compounds were examined for their ability to function as a neutral diluent and one compound, 2-hexadecyl-glycero-sn-3-phosphocholine (2H-GPC) was found to have the following properties. 2H-GPC formed micelles to which pig pancreatic PIA;! bound as demonstrated by fluorescence studies [25**]. In the prescence of a st&ently high concentration of 2H-GPC (typically 3mM), all of the PI& was bound to the interface. Next, the rate of alkylation of the active-site his&line residue (His48) by well known PIA inactivators, such as pni-
trophenacyl bromide, was studied. The rate of enzyme alkylation was found to be the same for the PIA;! either in aqueous solution or fully bound to 2H-GPC rnicelles. This result indicates that 2H-GPC does not occupy the catalytic site on E*, and that the binding of the akybing agent to the catalytic site and the binding of PI-42 to the interface are not synergistic [ 25”]. Thus, 2H-GPC has the properties of a neutral diluent and micelles of this compound provide a useful matrix with which to study the binding of other ligands to the catalytic site of E*. When the mole fraction of a ligand in the 2H-GPC interface is equal to the dissociation constant (in units of mole fraction) for the E*L complex (L, l&and), half the enzyme is present as E* and half as E*L Under these conditions, the half-time for alkylation in the presence of the ligand will be twice that measured in the absence of the ligand. This method of measuring lig and dissociation constants for PIA has been previously applied to the water-soluble form of the enzyme [211 and the extension to the study of E* as described here presents no major problems. Using this approach, the dissociation constants for a variety of ligand have been measured [25”]. For example, the K, for a potent enantiomer of a phosphate-containing transition-state analog was found to have a value of 0.001. This enantiomer, which has a glycerol backbone stereochemistry that is similar to that found in naturally occuring phospholipids (m-3) is a much better inhibitor than its enantiomer (sn1). Using this value of the Kt, a value of KM = 0.3 mole fraction for the DMPM substrate could be obtained from the inhibition data and equation (3). The KP for the combination of lipolysis products (fatty acid and lysophospholipid) was directly measured to be 0.025 using the protection method. In good agreement with this, a value of KP = 0.036 was obtained from the inhibition data with the transition-state analog in small vesicles using equation (4) together with the K, value of 0.001. Because the DMPM substrate undergoes catalytic tmnsformation, it is not possible to evaluate its interaction with E* using the protection method. However, in addition to dete rmining the Khl value from the inhibition data (see above), the value of this Michaelis constant could be obtained by applying the scooting-mode analysis to DMPM vesicles in the presence of the neutral diluent 2H-GPC was added to DMPM vesicles up to a mole fraction of -0.3 without solubilizing the vesicles. This provided a way of lowering the surface concentration (or mole fraction) of substrate that the bound enzyme ‘sees’. The resulting decrease in the initial enzymatic velocity that we observed could only have arisen from surface dilution because 2H-GPC does not function as a competitive inhibitor nor does it disrupt the vesicles when present up to a mole fraction of about 0.3. This analysis yielded a value of KM = 0.3 mole fraction [25**]. This number agrees well with the value of KM determined from the studies with several competitive inhibitors. It is interesting to note that although the PIA is embedded in a maximally packed array of DMPM substrate in the vesicle, the Khl for the substrate is not much lower than the substrate concentration (initially 1.0 mole fraction),
Quantitative
and structural
analysis
and the presence of trace amounts of potent inhibitors can lead to significant inhibition of the interfacial catalysis. This statement has obvious consequences for the design of medicinally important PLA2 inhibitors.
Structural studies of phospholipase inhibitor complexes
A,-
By using inhibitors of PLA2 with short alkyl chains, it has been possible to obtain crystals of three different enzyme-inhibitor complexes. Thunnissen et al. [28**] determined the crystal structure of the pig pancreatic PLk* in a complex with an inhibitor in which the enzyme-susceptible ester was replaced with an amide. The honey bee and Chinese cobra (Nuju nuju utru) venom PLA*s have been crystallized in the presence of a phosphonate-containing phospholipid analogue [29*=-31**]. The active-site region of the cobra venom structure is shown in Fig. 3. It can be seen that one of the inhibitor’s non-bridging phosphonate oxygens is bound to the Ca*+ ion and the other oxygen atom is hydrogenbonded to the active-site histidine residue. This result is consistent with the previously proposed mechanism for PIA2 catalysis [1,21] in which a histidine-bound water molecule attacks the carbonyf group of the substrate’s m2 ester to form a tetrahedral intermediate with the oxyanion of this intermediate bound to the Ca2+ ion. In addition, the amide NH of Gly30 donates a hydrogen bond to the oxyanion. The fact that the pro-S oxygen atom of the
of inhibitors
of phospholipase
A, Celb,
Berg
and
lain
inhibitor’s phosphate group is bound to the Ca2+ ion (Fig. 3) suggests that the cofactor helps to anchor the substrate in a catalytically productive manner in addition to its role in electrophilic catalysis. A tyrosine residue donates a hydrogen bond to the pro-R oxygen of the phosphate group in the cobra venom enzyme, whereas in the bee venom enzyme this function is fulhlled by a threonine residue. A detailed view of the Ca2+ ligation cage in the presence and absence of the inhibitor is shown in Fig. 4. In the uninhibited enzyme (Fig. 4a), the Ca2+ ion is heptacoordinate, with live ligands coming from the protein and two from solvent waters. In addition, a third water molecule is seen bound to the histidine residue. When the enzyme is bound to the phosphonate inhibitor, these three water molecules are replaced with inhibitor oxygen atoms with no detectable deformation of the protein structure (Fig. 4b). It is interesting to note that the constellation of active-site residues and the conformation of the bound inhibitor are virtually superimposable for the cobra- and bee-venom structures, despite the fact that the global structure of the bee-venom enzyme differs dramatically from the common structural architecture seen with other extracellular PIA2s. If it is imagined that the ester substrate binds to the enzyme with its carbonyf group liganded to the Ca2+ ion and with an attacking water molecule hydrogen bonded to the histidine, the ester would be required to rotate out of the plane defined by the trigonal carbonyl and the bridging oxygen. This can readily be seen from Fig. 3 by overlaying the ester onto the bound conforma-
.. & i
-
Fig. 3. Stereo-diagram of cobra venom metal ligations are indicated by dotted onto the phosphonate by least-squares enzyme structure.
phospholipase A, with a bound lines. Also shown is the structure superpositioning of the entire
phosphonate phospholipid analogue. The hydrogen bonds and of the bound amide inhibitor [27**1. The amide was overlayed pig pancreatic enzyme structure onto the entire cobra venom
841
842
Catalysis
and regulation
\
,
phospholipase A, in the absence of phosphonate inhibitor. The protein Fig. 4. (a) Coordination structure of the Ca 2+ ion in cobra venom donates five ligands to the metal coming from the amide carbonyl groups of residues 28, 30 and 32, as well as bidentate coordination from the side chain of Asp49. The three water molecules are denoted as W. (b) Coordination structure of the Ca2+ ion in the presence of the bound phosphonate phospholipid analogue. P2 denotes the phosphonate group and P3 denotes the phosphate group. The hydrogen bond between the oxygen of P3 and a tyrosine is not shown.
tion of the phosphonate. Such a rotation may be possible with the ester but would be prohibitively large for the amide. Indeed, the crystal structure of the enzymebound amide shows that the inhibitor is tilted in the active site relative to the phosphonate [28**]. This can be seen in Fig. 3 where the amide structure has been overlaid onto the bound phosphonate. The amide carbonyl group is bound to the Ca*+ ion and the amide NH is hydrogen-bonded to histidine. This later interaction leads to the displacement of the water molecule that is hydrogen-bonded to the histidine in the uninhibited enzyme. This hydrogen bond may also explain why amidecontaining phospholipid analogues bind 100-1000 times more tightly to the enzyme than the corresponding esters [32-l. Based on the fact that the structures of the enzymes in the presence and absence of the bound inhibitor are virtually superimposable on each other [ 29**], it is tempting to propose that a sign&ant conformation change does not occur in the enzyme upon binding to the lipid interface. It is clear from these crystal structures that a phospholipid molecule must diffuse from the plane of the membrane bilayer through a hydrophobic channel in the enzyme that contains the catalytic machinery. It is possible that there is a tight seal formed between the substrate interface and the Wet-facial recognition surface of the enzyme. It has been shown that the binding of the enzyme to the interface occurs with desolvation of the protein and lipid surfaces that contact each other [33]. This desolvation may allow the phospholipid to be transferred down the
hydrophobic channel with minimal contact with bulk solvent.
References
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GHOMASHCHI
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WASHBURNWH, DENNIS EA: Suicide-inhibitor BifunctionaBy Linked Substrates (SIBLINKS) as Phospholipase Aa Inhibitors. / Biol Cbem 1991, 266:5042-5048. REYNOIDSLJ, MORGAN BP, Hm Gq M~HEUCH ED, DE= W: PhosphoIipase 4 Inhibition and ModRication by Monoalogue. J Am Ckm SCX 1988, 110:5172-5177. GHOMASHCHI F, Yu B-Z, JAIN MK, GEIB MH: Kinetic Characterization of PhosphoIipase A2 ModIEed by Manoalogue. 1991,
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J.w MK, YUAN W, GELB MH: Competitive Inhibition of Pbospholipase A2 in Vesicles. Bfbcbemm 1989, 28:4135-4139. JALN MK, JAHAGUU%R DV: Action of Phospholipase A2 on BiIayers. ERect of Inhibitors. Biocbim Bi@ys Acfa 1985, 814:31+326.
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THUNNISSEN MMGM, A6 E, KU KH, DRENIH J, DIG KULPERS OP, DIJK~~AN R, DE m GH, VERHEIJ HM:
phospholipid bound as to why the amide 29. ..
Scorr PB:
DI
to an enzyme. phospholipid
The structure provides analogues bind tightly
WH~?E SP, Owt~ou%~
Z, YUAN W, GELB MH, SIGNER
Aa. Science 1990, 250:1541-1546. This series of papers [28**-30**] describes the first crystal structures of a phospholipid bound to an enzyme. The strucNm provide strong evidence for a catalytic mechanism for the esterolysis. 30. ..
SCOTTDL, O~INOWSKI Z, GELB MH, SIGNER PB: Crystal Struchue of Bee-venom PhosphoIipase A2 In a Complex with a Transition-state AnaIogue. Science 1990, 250:15631566.
This series of papers [28**-30**] describes the first crystal strucNres of a phospholipid bound to an enzyme. The structures provide strong evidence for a catalytic mechanism for the esteroh&. 31. ..
WHITT
SP, Scorr
DI
C&wt~owsto
Z, GELE MH,
This series of papers [28**-30**] describes the Iirst ctystaJ StrucNre of a phospholipid bound to an enzyme. The structures provide strong evidence for a catalytic mechanism for the esterolysis. RANSAC s, RMERE c, HAAS GH: Competitive
Soutt~ JM, G.wcm C, VERGER R, DE btbibition of Lipolytic Enzymes. 1. A
This paper discusses the theory for competitive inhibition mixed micelIes. The other papers that follow in the series amples of inhibitors and their analysis in mixed micelles.
JJ,
PIETERSON
WA,
DE
the Active Site of PhosphoIipase E&1446-1454.
HAAS
GH:
Histidine
AZ Biabemishy
at 1974,
SIGIER PB:
CyrstaI Structure of Cobra-venom Phospholipase A2 in a Complex with a Transition-state AnaIogue. Science 1990, 250:1560-1563.
BERGOG, Yu B-Z, ROGERS Jq JAIN MK: Interfacial Catalysis by Phospholipase Aa: Determination of the Interfacial Kinetic Rate Constants. Biochemistry 1991, 30:72837297.
VOLWERK
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Interfacial CataIysis: the Mechanism of Phospholipase
32. ..
accomplished for an enzyme that operates in an interface.
BW, X-ray
Structure of Phospholipase A2 Complexed with a Substratederived Inhibitor. Nuhtre 1990, 347689691. This paper and [29**,30**] describe the first crystal structures of a
Catalysis by Phospholipase Aa: Evaluation of the Interfacial Rate Constants by Steady-State Isotope Effect Studies. Bichzbemisf?y 1991, 30:7298-7305.
This paper develops the basic concept of scooting kinetics for interfacial catalysis on vesicles. Both the experimentaJ verification of scooting and the theoretical description of the process are detailed. Data from this paper and from papers that follow it in the series are puked together in order to extract most of the interfacial kinetic rate constants for the steps shown in Fig. 2. This is the first time that this has been 21.
27.
JAM MK. ROGER
%0:5162. 15.
A2 Gelb, Berg and Jain
This paper describes the theory and experimental observations of competitive inhibition in the scooting mode on both large and small vesicles. Also developed is the concept of a neutral diktent and the use of such a compound to measure the dissociation constants for a variety of ligands that interact with PL42 in the interface.
DC, NICHOLS JW: Characterization of PhosphoIipid Transfer between Mixed Phospholipid-Bile Salt Miceks. Biochem~ 1990, 29:87-. DA, SHOEMAKER
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..
2595734-5739.
FUUNGTON
of phospholipase
Biocbemishy 25.
11.
of inhibitors
Kinetic Model Applicable to Water-insoluble Competitive Inhibitors. Biochim Bic#&ys Acta 1990, 10435746.
33.
of PIA in provide ex-
Dehydration of the Lipid-Protein Microinterface on Binding of Phospholipase A2 to Lipid BIIayers.
J,UN MK, VAZ WIG: Biocbim
Bi@ys
Acta
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MH Gelb. Department of Chemistry, BG-10, University of Washmgton, Seattle, WA 98195, USA 0 Berg, Department of Molecular Biology, Uppsakt University, Sweden. hlK Jain, Department of Chemistrv and Biochemistry, University of Delaware 19716, USA
643