On the stereochemistry of catalysis by serine proteases

J. theor. Biol. (1974) 46, 543-558

On the Stereochemistry of Catalysis by Serine Proteases LAsz~6

POLG.~R AND BENCE ASB~TH

Enzymology Department, Institute of Biochemistry, Hungarian Academy of Sciences, Budapest, Hungary (Received 8 August 1973, and in revised form 20 September 1973) From stereochemical considerations and model building the following conclusions were drawn for the stereochemistry of the catalytic steps of chymotrypsin and subtilisin. (1) In contrast to previous stereochemical investigations, rotation of 120” or more of the oxygen atom of the “reactive” serine residue is not possible in the course of the reaction with specific substrates. (2) During catalysis the serine oxygen atom is approximately in the position found in the crystalline enzyme, i.e. at a distance of about 3 A from the nitrogen atom of the catalytically important histidine residue.(3) The detailedstereochemicalmechanisminvolves the formation of a strained tetrahedral intermediate and a strained acylenzyme.The strain energyis suppliedby the formation of a hydrogenbond betweenthe enzyme and a specificsubstrate.(4) The geometry of proton transfers in the intimate encounter complex of chymotrypsin is slightly but significantly different from that of subtilisin.

1. Introduction The main features of the mechanism of action of serine proteases shown in Fig. 1 were summarized earlier by Bender & Ktzdy (1965) and Blow & Steitz (1970). It is seen that a nucleophilic attack by the hydroxyl group of the “reactive” serine residue on the carbonyl carbon atom of the substrate is catalyzed by a histidine residue as a general base.This leadsto the formation of a tetrahedral intermediate and an imidazolium ion. The intermediate breaks down by general acid catalysis to an acyl-enzyme, an imidazole base Acylation

FIG. 1. Scheme of thereactionmechanism for serine proteases. Xstands for an OR’ or an NHR’ groupin acylationand for an OH groupin deacylation. 543

544

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POLGAR

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ASB6TH

and alcohol or amine. The acyl-enzyme is hydrolyzed via the reverse mechanism of acylation, hence acylation and deacylation have been regarded as symmetrical processes (Bruice, 1961; Bender, 1962; cf. Polgar, 19726). Of course, in the hydrolysis the hydroxyl group of a water molecule is the nucleophile instead of the hydroxyl group of the serine residue. The peculiarity of this catalysis is that the two subsequent proton transfers, general base and general acid catalyses, take place at one encounter of the reacting groups (Polgar, 1971; 1972~). This implies the formation of an intimate pair involving the tetrahedral intermediate and the imidazolium ion, and this intimate pair is stabilized by an extended hydrogen bond network. Since a one-encounter reaction of this type should diminish the entropy changes by restricting translational and rotational movements of the reacting groups in both acylation and deacylation, it is presumably an important source of the catalytic power of serine proteases. The possibility of formation of an intimate pair has not been considered in the previous studies on the stereochemistry of the catalysis by cr-chymotrypsin (Henderson, 1970; Fersht, Blow & Fastrez, 1973) and subtilisin (Wright, 1972; Robertus et al., 1972; Robertus, Kraut, Alden & Birktoft, 19726). In this paper the stereochemistry of the two linked proton transfers within the intimate encounter complex, which implies a shuttle mechanism, will be investigated. 2. Made1 Building The active site regions of cc-chymotrypsin and subtilisin were built from Kendrew model (Cambridge Repetition Engineers, Greens Road, Cambridge, England), on the basis of atomic co-ordinates given by Birktoft & Blow (1972) and Alden, Birktoft, Kraut, Robertus & Wright (1971), respectively. For designation of atoms, the IUPAC-IUB conventions (1970) were followed. As to the limiting distance between non-bonded atoms, the data of Ramachandran & Sasisekharan (1968) were employed. In studying the stereochemistry of catalysis we set out from the following considerations. (1) The intermediates to be identified are as follows: in acylation the enzyme-substrate or Michaelis complex, the tetrahedral intermediate and the acyl-enzyme; in deacylation the acyl-enzyme attacked by a water molecule and the tetrahedral intermediate (cf. Bender & Kezdy, 1965). (2) It follows from the symmetry postulate (Bruice, 1961; Bender, 1962) that the geometry of the tetrahedral intermediate in acylation and in deacylation is similar. (3) The atoms between which proton transfer takes place cannot be farther from each other than a hydrogen bond distance otherwise the energy

STEREOCHEMISTRY

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SERINE

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ACTION

545

barrier of the proton transfer would be too high. Therefore, in the tetrahedral intermediate the serine Oy, as well as the nitrogen or oxygen atom of the leaving group in acylation or the water oxygen in deacylation, cannot be farther from the NE2-H group of the imidazole ring than the van der Waals distance (- 3.4 A). This is a significant restriction in building the model, as compared to the criteria used in previous stereochemical studies (Henderson, 1970; Robertus et al., 19723). (4) The negative oxygen atom of the tetrahedral intermediate is stabilized by two hydrogen bonds from Z, and Z, groups of the enzyme (PolgPr, 1972u). In chymotrypsin these groups were identified as the backbone -NHgroups of Ser-195 and Gly-193 (Henderson, 1970) and in subtilisin as the -NHgroup of Ser-221 and the amide group of Asn-155 (Wright, 1972; Robertus et al., 1972u). Accordingly, we expect that hydrogen bond formation from Z, and Z, groups to the negative oxygen atom of the tetrahedral intermediate should be possible in the model. (5) Hydrogen bond formation between the -NHgroup of an amino acid derivative or peptide substrate and some group of the enzyme should be possible (Ingles & Knowles, 1968). This group was identified as the backbone carbonyl group of Ser-214 in chymotrypsin (Steitz, Henderson & Blow, 1969) and of Ser-125 in subtilisin (Wright, 1972; Robertus et al., 1972u). This interaction is referred to as the SiPi hydrogen bond (Schechter & Berger, 1967; Robertus et al., 1972a). (6) In the acyl-enzyme the carbonyl group of a specific substrate should be in a position where hydrogen bonding between the carbonyl oxygen and NC2 of the imidazole ring through a water molecule (CO. . . HOH. . . N”‘) is not possible. Otherwise the hydrolytic step would be inhibited as in indoleacryloyl-chymotrypsin (Henderson, 1970). An approximately perpendicular attack on the trigonal carbon atom of the substrate by the oxygen atom of the water molecule which is hydrogen bonded to N”’ would be feasible. The above considerations allow one to locate the tetrahedral intermediate more precisely than the Michaelis complex or the acyl-enzyme since the number and strength of interactions between enzyme and substrate are greater in the tetrahedral intermediate than in the other two intermediates. Therefore, the tetrahedral intermediates were built first and the Michaelis complexes as well as the acyl-enzymes were derived from them according to the above considerations. 3. Chymotrypsin (A)

ON

THE

ROTATION

OF

0’

DURING

CATALYSIS

Two types of orientations were found by X-ray diffraction studies for Oy of Ser-195 in a-chymotrypsin (cf. Birktoft & Blow, 1972): one in the

546

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ASBOTH

free enzyme hereafter called position A and the other in indoleacryloyl(Henderson, 1970) and tosyl- (Matthews, Sigler, Henderson h Blow, 1967) chymotrypsins hereafter called position B. Position B corresponds to the “down” position of Oy (cf. Henderson, 1970; Birktoft & Blow, 1972). It was suggested that Oy of Ser-195 is in position A in the active form of chymotrypsin and during acylation it rotates about the serine C”--Cfi bond by about 120” to position B (Henderson, 1970). It should be emphasized that position B was observed with tosyl- and indoleacryloylchrymotrypsins which can be regarded as inhibited rather than normal acyl-enzymes. Therefore conclusive evidences for the rotation of Oy with specific substrates cannot be obtained from the available X-ray diffraction data alone. In the following the implications of the rotation of Oy will be examined. Rotation of Oy away from the imidazole ring not only lengthens the hydrogen bond between Oy and N”’ (cf. Fersht, Blow & Fastrez, 1973), but completely disrupts it since the distance between the two atoms increases to about 4.4 A and at a distance greater than 34-3.5 A hydrogen bond formation is not possible (see also Discussion). Therefore proton transfer between Oy and NE2, which are as far as 4.4 A from each other, cannot take place as a concerted reaction with bond making and bond breaking. In fact, the idea of this rotation cannot explain “when the proton moves from the Ser-His hydrogen bond, when it approaches the nitrogen of the substrate and when the serine oxygen rotates to form a covalent bond with the substrate” (Henderson, 1970). If the idea of rotation is accepted, general base catalysis in acylation and general acid catalysis in deacylation required by experimental evidences (cf. Bender 8c KCzdy, 1965), should be rejected since general catalyses cannot take place concurrently with bond formation or bond breaking. Another possibility is that a rotation of more thait 120” takes place and 07 arrives at a position B nearer to NE2 where proton transfer might occur. This position is closer to that found in indoleacryloyl- and tosyl-chymotrypsins for Oy. However, such a rotation which implies an increase followed by a decrease of the distance between the reacting atoms in the same catalytic step would not be reasonable for chemical reasons. A further evidence against rotation is based on pure stereochemical grounds. Namely, en route to position B, Oy approaches the -NHgroup of Ser-195 to a distance of 2.5 A, which is significantly shorter than the extreme limiting distance. This imposes a considerable energy barrier on rotation. The other possibility to move Oy between positions A and B would be a rotation towards the imidazole ring. This is a movement opposite to that anticipated for Oy when it attacks the carbonyl carbon atom of the substrate

STEREOCHEMISTRY

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in the acylation step. Moreover, the rotation towards the histidine residue is inhibited sterically by the imidazole ring both in acylation and in deacylation. This is the case even if the imidazole ring is slightly displaced, which is stereochemically allowed. Accordingly we conclude that rotation of Oy of Ser-195 in chymotrypsin is not likely to occur during catalysis but rather the entire catalytic process takes place either in position A, or in position B which is as near to NE2 as possible. The detailed analysis which takes considerations l-6 into account will show below that catalysis is possible only in position A. (B)

GEOMETRY

OF

THE

CATALYTIC

INTERMEDIATES

For studying the mechanism of action of chymotrypsin, N-formyl-Ltryptophan amide was chosen as substrate. The position of the free acid in the complex with chymotrypsin is known from X-ray diffraction measurements (Steitz et al., 1969). In the course of model building we have found that the imidazole ring of His-57 is in an unfavourable position for the proton shuttle since the lone pair of electrons of Ne2 does not point between Oy and the nitrogen atom of the leaving group. In order to obtain a more favourable geometry we moved the imidazole ring by about O-5 A towards the interior of the enzyme. This movement involved a rotation of about 15” about the Ca-CB bond of His-57 and a small tilt of about 10” of the plane of the ring. Such a movement is not prevented by steric hindrance. Although Nd’ of His-57 approached CB of Ala-55 as compared to the position found in the crystalline enzyme but the distance between them (3.4 A) is still greater than the limiting distance (3.2 A). In the new position the Nd’-H group of His-57 forms a bifurcated hydrogen bond with Od’ and Od2 of Asp-102. This geometry is similar to what was found with crystalline subtilisin (Wright, 1972) and with crystalline trypsin (Krieger, Kay & Stroud, 1974) as well. In the new position the direction of the lone pair of electrons of NE2 is much more favorable for the proton shuttle both in positions B and A. (i) Tetrahedral intermediate The tetrahedral intermediate can be readily constructed in position A, but not in position B if the above chemical considerations are taken into account. The position of the indole ring of the substrate in the hydrophobic pocket in position A is similar to that found in the formyl-L-tryptophan-chymotrypsin complex (Steitz et al., 1969); in position B it is similar to that found with indoleacryloyl-chymotrypsin (Henderson, 1970). These two positions of the indole ring change only slightly during catalysis.

548

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Fro. 2. Geometry of proton shuttle in positions A and B in chymotrypsin. Naa lies in the “yz” projection plane. Tetrahedral intermediate in position A is drawn in heavy lines, in position B in dotted lines. The heights (A) of some atoms relative to this plane are indicated in brackets. The arrow shows the direction of the Naa-H bond.

The negative oxygen of the tetrahedral intermediate is bound by two hydrogen bonds to the backbone -NHgroups of Ser-195 and Gly-193 in position A; in position B, a hydrogen bond can be formed only with Ser-195. The most important differences between positions A and B are seen in Fig. 2 (tetrahedral intermediate in position A is shown by heavy lines and in position B by dotted lines). In position A the distances from Ne2 to Oy and to the oxygen or nitrogen atom of the leaving group are 2.9 and 3.1 A, respectively. These distances are suitable for hydrogen bonds. In position B the distance between Oy and Ne2 cannot be smaller than about 4 A, because a shorter distance would imply an abnormally short SIP, hydrogen bond. Furthermore, the direction of the Ne2-H bond (shown by an arrow in Fig. 2) is more favorable in position A than in position B for the proton shuttle. (ii) Michaelis complex The Michaelis complex is derived from the tetrahedral intermediate by moving the carbonyl carbon atom of the substrate from the covalent bond distance to about 2.7 A away from Oy, which is the limiting distance for non-bonded oxygen and carbon atoms. Furthermore, the plane of the trigonal carbon atom was positioned to be approximately perpendicular to the direction of the attack by 0”. A significant difference between the two Michaelis complexes is that in position B there is a strong S,P, hydrogen bond (2.8 A), whereas in position A this distance is 3.4 A, too long for a normal hydrogen bond.

STEREOCHEMISTRY

OF SERINE

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549

(iii) Acyl-enzyme When the acyl-enzyme is formed from the tetrahedral intermediate the carbonyl oxygen turns towards the solvent. The plane of the trigonal carbon atom is about perpendicular to that occupied in the Michaelis complex. Therefore, hydrogen bonds from the backbone -NHgroups of Ser-195 and Gly-193 to the carbonyl oxygen cannot be formed in position B; in position A a weak hydrogen bond might be formed with Gly-193. An important difference between positions A and B is that non-productive bridging of the carbonyl oxygen and Ne2 by a solvent water molecule, like in indoleacryloyl-chymotrypsin (Henderson, 1970), is impossible in position A whereas it is certainly allowed in position B. Accordingly we conclude that with specific amino acid derivatives the catalysis can proceed only in position A. 4. Subtilisin

To study the stereochemistry of the mechanism of action of subtilisin we have chosen acetyl-L-alanyl-glycyl-r.-phenylalanine amide since one can identify the approximate binding mode of this substrate in light of the binding of its inhibitory chloromethyl ketone derivative (Robertus et al., 1972a). Of course, the exact positions of the reacting atoms during catalysis do not coincide with those of the inhibitor. (A) TETRAHEDRAL

INTERMEDIATE

The tetrahedral intermediate can be built without moving the side chains of Ser-221 and His-64 from their positions found in the crystalline enzyme.

C N 10

FIG. 3. Intims ion in subtilisin relative : to this N’rH

:ounter complex of the tetrahedral intern mediate and the lies in the “yz” projection plane. The heights (A) of some atoms ! are indicated in brackets. The arrow shows the direction of the

550

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ASB6TH

The phenylalanine side chain is embedded in the S, crevice as found with the chloromethyl ketone derivative (Robertus ef al., 1972a). The length (about 2.9 A) and direction of S,P, hydrogen bond correspond to a strong hydrogen-bonding interaction. Hydrogen bond distances to the oxyanion from the backbone -NHgroup of Ser-221 and from the -NH, group of Asn-155 are 2.8 and 3.2 A, respectively. The nitrogen atom of the leaving group is at a distance of 2.9 A from NE2 of His-64 (Fig. 3). The lone pair of NE2 points between Oy of Ser-221 and the leaving atom. It is about equidistant from both atoms, in contrast to the case of chymotrypsin. (B)

MICHAELIS

COMPLEX

The Michaelis complex was built on the basis of the same principle used with chymotrypsin. S,P, hydrogen bond is certainly non-existent in the subtilisin complex since the distance between the oxygen and nitrogen atoms is 3.6 A. Hydrogen bond formation, however, is possible between the carbonyl oxygen atom of the substrate and -NH2 group of Asn-155 since the bond is 2.7 A long and the direction is also suitable. (C)

ACYL-ENZYME

The acyl-enzyme was also derived from the tetrahedral intermediate. The position of the phenylalanine side chain does not change. SrP, hydrogen bond length slightly increases (to about 3.1 A). The carbonyl oxygen of the substrate approaches -NH2 of Asn-155 to about 2.8 A, while the interaction with the -NHgroup of Ser-221 is abolished. The position of the trigonal carbon atom is suitable for the general base catalyzed attack by a water molecule in the deacylation step. Non-productive binding (CO. . . H-O-H. . . Ne2) seems to be impossible because of the relative position of the groups and the strong hydrogen bond from Asn-155 to the carbonyl oxygen. 5. Strain in the Catalytic Intermediates When the tetrahedral intermediate is formed with a specific amino acid derivative, the new covalent bond will be strained. This strain is due to the short distances between CB of the reactive serine residue and C” and CB of the substrate (Fig. 4). The importance of strain in enzymatic catalysis has earlier been suggested (Lumry, 1959; Jencks, 1963). Experimental evidence for the occurrence of strain can be found in the catalysis by lysozyme where the ring of N-acetylmuramic acid is forced into a strained “half-chair” conformation (Rupley & Gates, 1967). The strain in the covalent intermediates of serine proteases

STEREOCHEMISTRY

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551

FIG. 4. Strained conformation in the tetrahedral intermediate. Angle COyCB is 114” 47; bond lengths for Cfl-07, 07-C and GCY bonds are 1 &I 1 a33 and 1 a54 A, respectively (Curl, 1959). Angle CXOY is 109.5’; dihedral angle 0 (C”COX?) is -25”.

is similar to that found with the eclipsed form (dihedral angle = 0”) of butane. The energy content of this conformation is by about 5 kcal/mole greater than that of the minimum energy form (Hendrickson, 1967). (A)

CALCULATION

OF

THE

APPROXIMATE

STRAIN

ENERGY

In calculating the strain energy there are three kinds of interactions to be considered. (I) The C.. .C, C.. .H and H.. .H non-bonded interactions between CBH, of serine and C”, @H, of the substrate (see Fig. 4, hydrogens are not shown). (2) The bond angle distortion or bending energy resulting from the distortion of bond angles C’COy and C07C? (Fig. 4). (3) The torsional strain which depends on the dihedral angle (Fig. 4). These energies are interdependent, so that, for example, at constant dihedral angle by increasing the C”COy and COY@ bond angles interatomic distances will become greater and thus repulsion between non-bonded atoms decreases. As the bending force constant does not differ too much for bond angles of different groups (Ramachandran t Sasisekharan, 1%8), in our calculations we assumed that the CaCOy and COyC” bond angles are distorted to the same extent. The above three kinds of energy are shown in Fig. 5, each as a function of the bond angle distortion, in the case of the tetrahedral intermediate. The torsional energy was calculated at a constant dihedral angle of about -25” as estimated from the model. The sum of these energies has a minimum at about 9” bond angle distortion (Fig. 5). This means that both COyC@ and C”COy bond angles are distorted by 9”. The value of this minimum, i.e. the strain energy of the tetrahedral intermediate, is about 5 kcal/mole. The strain energy of the acyl-enzyme was calculated similarly. However, there are two significant differences: (1) the C”COy bond angle is about 115” (Dunitz & Strickler, 1968) instead of 109.5” used in the tetrahedral intermediate; (2) the minimum energy conformation of the esters is planar and T.B. 36

552

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AND Interatomic

2.6 I i2

2.8 I

I

B.

ASB6TH

distance

(8)

I

3.0 I

I

3.; I

8

IO

I2 12

I4 14

.

2

4 Bond

6 angle

distortion

(degree)

FIG. 5. Strain energies of the tetrahedral intermediate as functions of bond angle distortion and the corresponding distances from C4 of serine to c” and CY of the substrate. Curve x-x-x stands for the energy of non-bonding interactions calculated by the aid of Buckingham type function, using the constants given by Hendrickson (1967). Curve o-o-o represents the bond angle distortion energy calculated according to Ramachandran & Sasisekharan (1968). It is the energy required if the angles COV and c”COY are both distorted by the value shown on the abscissa. The dotted line shows the torsional energy calculated according to Ramachandran & Sasisekharan (1968). The sum of these energies (a) represents the total conformational energy of the tetrahedral intermediate.

an out-of-plane distortion would require high energy. This implies that the dihedral angle 8 (CaCOyCa) in the acyl-enzyme should approach 0”. According to the above reasoning, the strain energy of acyl-enzyme was calculated at 0” dihedral angle. We obtained 15 and 1.6 k&/mole energies for the non-bonded interactions and bending, respectively, at the minimum energy conformation. Because of the partial double bond character of the C-0’ bond in the acyl-enzyme, we calculated the torsional energy in a way different from that used with the tetrahedral intermediate. As an approximation, we utilized the data on the analogous conformation of ethyl formate, according to which the conformationa energy is 2.5 kcal/mole (Tabuchi, 1958). We assumed that the bond angles are not distorted at this minimum energy conformation. The energy of the non-bonded interactions in this conformation of ethyl formate was calculated to be 1.8 kcal/mole, which was subtracted from the total conformational energy. Thus we obtained O-7 k&/mole for the torsional energy in ethy1 formate and used this

STEREOCHEMISTRY

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553

ACTION

0.7 kcal/mole in the case of the acyl-enzyme, too. Thus, by summing the three components we obtained about 3.8 k&/mole strain energy for the acylcnzyme, which does not differ appreciably from that found with the tetrahedral intermediate. (B)

SIPl

HYDROGEN

BOND

AS

A SOURCE

OF

THE

STRAIN

ENERGY

It is clear from model building that the formation of the SiPi hydrogen bond runs parallel with the formation of the tetrahedral intermediate. Thus it is reasonable to assume that the strain energy is supplied by this hydrogen bond. In fact, the energy of the S,P, hydrogen bond is certainly greater than that calculated for the strain. In general, hydrogen bonding energy is 3-8 kcal/mole (Ramachandran & Sasisekharan, 1968). On the basis of recent studies on hydrogen bond formation with formamide (Johansson & Kollmann, 1972) one can take the energy of a hydrogen bond between peptide groups as 6-8 kcal/mole, if the direction and the length of the bond are optimal. In acylation the tetrahedral intermediate is formed from an unstrained Michaelis complex, thereafter it contains about 5 kcal/mole strain energy. In deacylation the tetrahedral intermediate is formed from the acyl-enzyme, which already contains considerable strain energy (4 kcal/mole) and thus the energy barrier for the formation of the tetrahedral intermediate is much lower in this step. Accordingly, as a source of the strain energy, the Sip, hydrogen bond should be more important in acylation than in deacylation. This implies that specific substrates, which are capable of forming the S,P, hydrogen bond should be much better substrates than their analogues lacking the -NHgroup, and that the relative rate with specific substrates as compared to the analogues is expected to be higher in acylation than in deacylation, provided that other factors do not substantially interfere with catalysis. The data compiled in Table 1 strongly support this assumption. The acylation (/Q/K,) and deacylation (k3) rate constants found in the literature were normalized for inherent reactivity differences in the three substrates. As correction factors for iV-acetyl+phenylalanyl, 0-acetyl-r-j?-phenyl-lactyl and j?-phenylpropionyl derivatives the reciprocals of the rate constants of alkaline hydrolysis of ethyl N-acetyl-glycinate, ethyl 0-acetyl-glycolate and ethyl acetate, respectively, were used. The rate constant of the base-catalyzed hydrolysis of ethyl acetate was taken to be O-148 M-’ set-’ at 25” (Polgiir, 197B) and the corresponding rate constants of ethyl N-acetyl-glycinate and ethyl 0-acetyl-glycolate were l-31 and 4.1 M-I set-‘, respectively, according to our measurements. Inspection of the corrected values in Table 1 shows that in the case of the ethyl N-acetyl+phenylalaninate the rate constant in

554

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POLGhR

B. ASB6TH

AND

TABLE

1

Chymotrypsin-catalyzed hydrolysis of ethyl estersof N-acetyl+phenyIalanine and its analogues Substrates Ethyl N-acetylL-phenylalaninate Ethyl O-acetylL+phenyllactate Ethyl bphenylpropionate

Acylation kalK WK,ccrr

ka II

Deacylation ka corr

63oooT

48ooo

94.8

72.2

262

6.3

9.2

2.2

7.28

48.6

0.074

030

As correction factors for the ethyl esters of N-acetyl-L-phenylalanine, 0-acetyl-L-bphenyllactic acid and fiphenyl-propionic acid the reciprocals of the rate constants of alkaline hydrolysis of the ethyl esters of N-acetyl-glycine, O-acetyl-glycolic acid and acetic acid, respectively, were used. t Data of Hammond & Gutfreund (1955). $ Data of Cohen & Weinstein (1964). 8 Data of Laidler & Barnard (1967). 11Data of Ingles & Knowles (1968).

acylation is higher by 3-4 orders of magnitude and in deacylation by about two orders of magnitude as compared to the analogous compounds unable to form the SIPI hydrogen bond. The calculated strain energy can provide such an enhancement of the rate constants. 6. Geometry of Proton Transfers in Chymotrypsin and Subtilisin

Although the evolution of chymotrypsin and subtilisin took place in entirely different ways, the active site regions are very similar. The positions of 27 atoms of the catalytically important residues agree within an average error of O-8 A (Robertus et al., 1972a), which accounts for the common mechanism of the two enzymes. However, as seen from Figs 2 and 3, the geometries of the two shuttles in the two proteases are remarkably different and we believe that this difference is greater than the experimental error of model building. Figure 2 shows that in chymotrypsin the two proton transfers occur approximately in the plane of the imidazole ring whereas in subtilisin (Fig. 3) the proton transfers take place towards and from the plane of the ring since the attacking and the leaving atoms of the tetrahedral intermediate are situated on the two sides of the plane of the imidazole ring.

STEREOCHEMISTRY

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555

Furthermore, it can be seen in Figs 2 and 3 that the distribution of the lone pair of NE’, that is the direction of the Ne2-H bond of the protonated histidine, is far less symmetrical in chymotrypsin than in subtilisin. In acylation the lone pair of electrons is closer to the leaving atom than to the attacking atom. The opposite is true in deacylation. A symmetrical distribution is possible neither in position A nor in position B of the tetrahedral intermediate. We cannot offer any convincing explanation as to the catalytic significance of this asymmetry. A priori the symmetrical distribution is expected to be more favourable. It should be kept in mind that the unfavourable direction of the hydrogen bond decreases only the stability and not the reactivity of the bond. Thus proton transfer may even be facilitated in chymotrypsin by the highly bent hydrogen bond (Ingraham, 1972). 7. Discussion

Our aim was to establish the stereochemistry of the elementary steps of catalysis by serine proteases. We have built the models of the catalytic intermediates by using X-ray diffraction data from the literature and chemical considerations listed above. One of the most important criteria of model building not considered in previous stereochemical studies (Henderson, 1970; Robertus et al., 1972a, 1972b; Fersht, Blow & Fastrez, 1973) is related to the geometry of the proton transfers to and from NE2 of the imidazole ring. This restricts the donor and acceptor atoms within van der Waals contact to NE2 in the intimate encounter complex of the tetrahedral intermediate and the imidazolium ion. We found that such a complex can be built for both chymotrypsin and subtilisin with all the considerations described above taken into account. The distances of about 3 A found between N” and the donor and acceptor atoms support our previous suggestion on the formation of a bifurcated hydrogen bond at the active centre (Polgar, 1971). This implies that the proton interacting with three atoms at the same time can form a covalent bond with any of them without marked changes in its position. The importance of hydrogen bond in proton transfers also follows from the reasoning of Hine (1972) according to which proton transfer should take place along a hydrogen bond. Furthermore, molecular orbital calculations show that the activation energy for proton transfer between two oxygen or two nitrogen atoms located at hydrogen bond distance from each other (2.7 and 2.8 A, respectively) rises by about 7 kcal/mole on increasing the distance between them by 0.1 A (Ingraham, 1972). Thus in position B proton transfers cannot take place, because the distance between Or and Ne2 is about 4 A and Oy cannot rotate towards NL2 during catalysis as it was pointed out above.

556

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POLGAR

AND

B.

ASB6TH

The tetrahedral intermediate is strongly stabilized by a number of interactions with the protein. Thus, the position of C of the substrate (cf. Fig. 2) is determined by the binding of the aromatic ring and the S,P, hydrogen bond. The negative oxygen atom is positioned in the oxyanion hole. The position of Oy is defined by the covalent bond and the interaction with N”. The leaving and attacking atoms in acylation and deacylation, respectively, also interact with N”‘. The many restrictions imposed on positioning of the tetrahedral intermediate by the above criteria permitted us to build the model of this intermediate about as precisely as that of the protein itself. The position of the acyl-enzyme is less accurate, and the location of substrate in the Michaelis complex is even more ambiguous. Namely, the position of the substrate in the Michaelis complex has been derived from the structure of the tetrahedral intermediate by moving the carbonyl carbon from Oy of the serine residue approximately along the covalent bond to be cleaved to a distance of 2.7 A. This movement, however, may be affected by interaction between the rest of the substrate and the protein, and it is difficult to estimate as to what extent. In previous models (Henderson, 1970; Robertus et al., 19726) the Michaelis complex was of great importance since it seemed to be identified on the basis of X-ray crystallographic measurements with inhibitors or products. It should be borne in mind, however, that the positions of the catalytic groups of an enzyme may be slightly different during catalysis and in the crystalline form. Therefore, from such X-ray crystallographic data one can only infer approximately as to the true Michaelis complex since the covalent bond (with chloromethyl ketone inhibitors) or the electrostatic interaction between the carboxylate ion and the protonated imidazole group (with products) is the major factor that determines the binding of the molecule. Likewise, one cannot infer from the geometry of an acyl-enzyme formed with a poor substrate to the correct position of the chemical bond to be altered in an elementary step with a specific substrate. In our opinion the crux of the matter in delineating the stereochemistry of serine protease catalysis is to adopt the intermediate defined the best and from this to deduce the other intermediates. As the available X-ray data were deemed by us to be insufficient to define the position of Michaelis complex or acyl-enzyme, the chemical considerations pertinent to the structure of the tetrahedral intermediate were given a predominant role. This led us to modify, within stereochemical possibilities, the position of the imidazole ring of His-57 found in crystalline chymotrypsin and to rule out position B which was actually revealed by X-ray diffraction. In spite of the uncertainties it can be stated with confidence from the model that a significant S,P, hydrogen bond cannot be present in the Michaelis

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ACTION

557

complex. This is important since the formation of such a bond may compensate for the energy requirements of strain. The energetic contribution of S,P1 hydrogen bond to deacylation was estimated as nearly 4 kcal/mole by Ingles & Knowles (1968). Their estimation was based on the great difference in the rate of deacylation of the enantiomers of N-acetyl-phenylalanyl-chymotrypsin (kf;lky = 6400). However, this does not reflect the proper contribution of the hydrogen bond to deacylation. Namely, in the case of the D-enantiomer unfavourable interactions occur between the enzyme and the substrate as indicated by the decreased deacylation rate of N-acetylo-phenylalanyl-chymotrypsin relative to that of /&phenylpropionyl-chymotrypsin. The corrected deacylation rates for these two acylchymotrypsins are O*Oll and O-48, respectively, calculated from the data of Ingles & Knowles (1968). As seen from Table 1, the contribution of S,P, is much more significant in acylation than in deacylation. Since the S,P, hydrogen bond affects both acylation and deacylation, Ingles & Knowles (1968) suggest that the hydrogen bond has already been present in the Michaelis complex and its significance lies in ensuring a high energy conformation of the substrate. Our model building experiments and that of Robertus et al. (1972) are not consistent with the formation of this bond in the Michaelis complex. Robertus et al. (19726) suggest that this hydrogen bond plays a role in the stabilization of the transition states. However, electronic effects on the tetrahedral intermediate, which influence activation energy in the first place should come from the extended hydrogen bond network, i.e. from interactions with the 2, and 2, groups as well as with the imidazolium ion (Polgar, 1972u), rather than from the distant SIP, hydrogen bond. As to the stabilization of the position of the reacting atoms, S,P, cannot be essential since these positions are well defined by a number of other interactions, too, as pointed out above. If SIP1 hydrogen bond is formed in the tetrahedral intermediate, this accounts for its importance both in acylation and in deacylation. It is known (cf. Jencks, 1969) that binding of the substrate in the Michaelis complex may result in strain as found with lysozyme (Rupley & Gates, 1967). In a recent paper on leaving group specificity in the chymotrypsin-catalyzed hydrolysis of peptides Fersht et al. (1973) suggest the formation of a strained Michaelis complex as one alternative interpretation of their results. This strain is relieved when Oy rotates by about 120”. It appears to us that the Michaelis complex can be built in position A without any strain. Nevertheless, if strain occurs, this would help to expel the leaving group from the tetrahedral intermediate rather than force Oy to rotate. The strain in serine proteases discussed above in details is unique at present, inasmuch

558

L.

POLGhR

AND

B.

ASB6TH

as it is formed in a covalently bound intermediate and not in a Michaelis complex. However, it is expected that this type of strain is of general importance in enzymatic catalysis. REFERENCES ALDEN,

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(A. Rich & N. Davidson, eds) p. 595. San Francisco: Freeman and Co. FERSHT, A. R., Bww, D. M. & FASTREZ, J. (1973). Biochemistry, N. Y. 12, 2035. HAMMOND. B. R. & GIJTFREUND. H. (1955). Biochem. J. 61. 187. HENDER&, R. (1970). J. molec..BioL‘S4, 341. HENDRICKSON, J. B. (1967). J. Am. them. Sot. 89, 7036. HONE, J. (1972). J. Am. them. Sot. 94, 5766. INOLES, D. W. & KNOWLES, J. R. (1968). Biochem. J. 108, 561. INGRAHAM, L. L. (1972). Biochim. biophys. Acta. 279, 8. IUPAC-IUB Commission on Biochemical Nomenclature (1970). J. mofec. Biol. 52, 1. JENCKS, W. P. (1963). A. Rev. Biochem. 32, 639. JENCKS, W. P. (1969). Catalysis in Chemistry and Enzymology, p. 282. New York: McGrawHill. JOHANSSON, A. & KOLLMANN, P. A. (1972). J. Am. them. Sot. 94, 6196. KRIEGER, M., KAY, L. M. & STROUD, R. M. (1974). J. molec. Bill. 83,209. LAIDLER, K. J. & BARNARD, M. L. (1967). J. Am. them. Sot. 89, 1009. LUMRY, R. (1959). In The Enzymes(P. D. Boyer, H. Lardy & K. Myrbiick, eds) p. 157. New York: Academic Press. MA~EWS, B. W., SIGLER, P. B., HENDERSON, R. & BLOW, D. M. (1967). Nature, Land.

214, 652. POLGAR, L. (1971). J. theor. Biol. 31, 165. POLGAR, L. (1972~). Acta biochim. Biophys. Acad. Sci. Hung. 7, 29. POLGAR, L. (19726). Acta biochim. Biophys. Acad. Sci. Hung. 7, 319. RAMACHANDRAN, G. N. & SASI~EKHARAN, V. (1968). Adv. Protein. Chem. 23, 283. ROBERT~S, J. D., ALDEN, R. A., BIRKTOFT, J. J., KRAUT, J., POWERS, J. C. &WILCOX, P. E. (1972~). Biochemistry, N. Y. 11, 2439. ROBERTUS, J. D., KRAUT, J., ALDEN, R. A. & BIRKTOFT, J. J. (19726). Biochemistry, N. Y.

11,4293. RIJPLEY, J. A. & GATES, V. (1967). Proc. natn. Acad. Sci. U.S.A. S7, SCHECHT~R, I. & BERGER, A. (1967). Biochem. biaphys. Res. Cornman. STEITZ, T. A., HENDERSON, R. & BLOW, D. M. (1969). J. tnolec, Bill, TABUCHI, D. (1958). J. them. Phys. 28, 1014. WRIGHT, C. S. (1972). J. molec. Biol. 67, 151.

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