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Kinetic analysis of rat liver sorbitol dehydrogenase
Int. J. Biochern. Vol. 15, No. 5, pp. 651-656, 1983 Printed in Great Britain. All rights reserved
KINETIC
Department
0020-711X.83~050651-06$03.00;‘0 Copyright 0 1983 Pergamon Press Ltd
ANALYSIS OF RAT LIVER DEHYDROGENASE
SORBITOL
NANCY C. LEISSING* and EUGENE T. MCGUINNESS~ of Chemistry, Seton Hall University. South Orange, NJ 07079. U.S.A. (Rrceired
2 Augusf
1982)
Abstract-l, A steady state kinetic investigation was performed on an improved preparation of rat-liver sorbitol dehydrogenase (L-iditol: NAD-oxidoreductase, EC 1.1.1.14). 2. Data analyses indicate the enzyme follows a rapid equilibrium random mechanism in the direction of sorbitol oxidation and a random mechanism in the direction of fructose reduction. 3. Kinetic constants were: KEAD 0.082 mM; Kzrbi’“’ 0.38 mM; KiADH 67 pm; .k”““” 136 mM. 4. Evidence is adduced to indicate the more rapid reverse (fructose reduction) reaction is susceptible to metabolic control by formation of abortive enzymeefructose-NAD and enzyme-NADH-sorbitol com-
plexes.
INTRODUCTION
Comparative amino acid sequence studies demonstrate that the enzymes, sheep liver sorbitol dehydrogenase (SDH), horse liver alcohol dehydrogenase (LADH) and Drosophilia alcohol dehydrogenase (DADH) provide examples of dehydrogenases that have different substrates (ethanol and sorbitol) but are structurally related (LADH and SDH), and of dehydrogenases that utilize the same substrate (ethanol) though they (DADH and LADH) lack close structural relationships (Jeffery et al., 1981; Jornvall ef al., 1981). Similarly, Klebsiella ribitol dehydrogenase (RDH) is revealed to have a closer structural homology to DADH than to SDH. While rate studies on ADH continue to generate useful insight about its function (e.g. Tsai, 1982) comparable information on the polyol dehydrogenases is sparse or unavailable. Ribitol dehydrogenase from Aerobacter aerogenes, for example, was found to follow an ordered bi bi mechanism involving ribitol and D-ribulose as the inside reactant pair with the formation of two abortive ternary complexes (Fromm & Nelson, 1962). D-Mannitol dehydrogenase from Absi&a glauca was shown to follow an ordered mechanism which is uniquely susceptible to metabolic control by mannitol l-phosphate acting as a dead-end inhibitor (Ueng & McGuinness, 1977). Sorbitol dehydrogenase from sheep liver is reported to obey a rapid equilibrium random mechanism in both directions that includes the formation of an abortive sorbitolenzyme-NADH complex (Christensen et al., 197.5). Correlative studies of structure-function relationships the alcohol-polyol dehydrogenases will among depend on a better understanding of the kinetic behavior of enzymes in the latter category.
The present report deals with a steady state kinetic investigation of an improved preparation (Leissing & McGuinness, 1978) of rat-liver sorbitol dehydrogenase, first partially purified from this tissue by Blakley (1951). Data analysis reveals the presence of kinetically significant abortive ternary complexes in the direction of fructose reduction. It remains to be established whether this characteristic is a property unique to the liver enzyme or is also an attribute, and of metabolic significance, to lens sorbitol dehydrogenase (Jedziniak et al., 1981).
MATERIALS AND METHODS Sorbitol and fructose were obtained from Pfanstiehl Laboratories. Proteins and nucleotide cofactors were purchased from Sigma Chemical Co. Sephacryl S-200 was obtained from Pharmacia. Buffers, including Tris-HCI and HEPES (N-2-hydroxyethyl piperazine-N’-2-ethane sulfonic acid) were procured from Calbiochem-Behring Corp. Electrophoresis support media was purchased from Beckman Instruments, Inc. All other chemicals were reagent grade or of superior quality. Enzpe
preparation
Sorbitol dehydrogenase from rat liver was purified up to and including Step 3 as discussed elsewhere (Leissing & McGuinness, 1978). Purification Step 4 was performed using a Sephacryl G-200 column (2.6 x 25cm) previously equilibrated with 10mM phosphate buffer, pH 7.5, containing 1 mM DDT (dithiothreitol). Elution was carried out in an upward direction using a flow rate of about 60 ml/hr. Active fractions from several runs were pooled, divided into aliquots and stored at - 2o’C until used. The partially purified enzyme preparation was stable in this form for at least 6 months. This preparation was judged to be enzymically homogeneous based on the consistent relative rates of oxidation by various polyols for Steps 3-5 (Leissing & McGuinness. 1978). Protein and unz~me vmsurements
* Present address: Department of Safety Assessment, Travenol Laboratories, Morton Grove, IL 60053, U.S.A. t To whom correspondence and reprint requests should be addressed.
Protein measurement was carried out after precipitation with tricholoacetic acid using a Coomassie blue protein assay as described by Bradford (1976). Bovine serum 651
NANCY C. LEISSING and EUGENE T. MCGLJINNESS
652
albumin was used as the standard. Estimate of the enzyme content was performed with the Beckman Microzone Cell. Duostat and R-l 12/l 15 scanning densitometer using pH 8.6 barbital buffer and ponceau S dye with visualization of the single activity band by staining with sorbitol, NAD+, phcnazine methosulfate and nitroblue tetrazolium, according to Fine & Costello (1963).
Sorbitol dehydrogenase kinetic studies were performed using a Gilford 3500 Automatic Analyzer with General Kinetic Card No. 5. The rate of change of absorbance at 340nm was followed at 25 C. For the identification of enzyme location from gel chromatography tubes the assay system contained 1.3 mM NAD’, 7.3 mM sorbitol. 50 mM Tris+HCl buffer (pH 9.6) and 0.1 ml enzyme preparation in a total volume of 1.35 ml. For initial velocity and product inhibition studies in both the forward and reverse directions, the assay system contained 50 mM HEPES Butler (pH 7.5), ionic strength 0.1 with the NaCI. and up to 0.1 ml of the pooled Step 4 enzyme preparation in a total volume of 1.35 ml. All reaction progress curves were linear for several minutes for the concentrations of substrate used. Blank reactions containing all components except alternate substrate were used as controls in all studies. Each experimental run was carried out in duplicate. Data from preliminary initial velocity studies were evaluated by computer fitting to the equation
C’IIAI
” = K,+CAI
SO
0
r
12l
I
I
I
I/Sorbitol,
mM-’
(1)
using a weighted least-squares numeric coefficient calculation (Wilkinson. 1961). At the ligand concentrations used the errors were found to conform to a normal distribution with approximate equal variances. Subsequently, all initial-velocity data were analyzed using the appropriate rate equations and computer programs described by Cleland (1967. 1979). In addition. replots of slopes and intercepts were examined for linearity using the Statistical Analysis System (SAS) software package with the General Linear Model (GLM) procedure. When the u~.Y’ term (.v = a,, + U,Y + as_?) was significant at the P < 0.05 level. the replot was judged to be nonlinear. Product inhibition data were also evaluated using the model-testing (as opposed to model-fitting) approach, as discussed by Fromm (197.5) and Hocking & Leslie (1967), to verify the nature of each inhibition pattern.
RESULTS
I/NADH, mM-’
I
I
I
I
0.02
0.04
0.06
0.06
I /Fructose, These were carried out using NAD+ and sorbitol
I 6
4
2
in the forward direction (Fig. 1A) as the variable
substrates against sorbitol and NAD+ as the changing-fixed substrates, respectively. In the reverse direction, D-fructose and NADH were the variable substrates. respectively, with NADH and D-fructose (Fig. 1B) as the changing-fixed substrates. All double reciprocal primary plots were intersecting. All slope and intercept replots were linear as determined by the procedure described in Materials and Methods, except for the “concave downward” slope replots generated by the reactions with NAD+ and sorbitol (Fig. IA) as variable substrates. A summary of the computer calculated kinetic parameters from these initial velocity studies is shown in Table 1. Inhibition
I 0. IO
mM-’
Fig. 1A. Initial velocity double reciprocal plot with sorbitol as the variable and NAD+ as the changing fixed substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50mM HEPES, 0.10 ionic strength; 29 pg of enzyme; sorbitol (as shown) and NAD+ [0.05 mM (O), 0.08 mM (0). 0.10 mM (a), 0.14 mM (o), 0.25 mM (m), l.OOmM (A). (00) and (A--A) refer to slope and intercept replots. respectively. B. Initial velocity double reciprocal plot with fructose as the variable and NADH as the changing fixed substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50mM HEPES, 0.10 ionic strength; 29jig of enzyme; fructose (as shown) and NADH CO.022 mM (0). 0.027 mM (O), 0.035 mM (A). 0.048 mM (0). 0.077 mM (w)], (-0) and (A---A) refer to slope and intercept replots, respectively.
Sorbitol Table
1.
Kinetic
KN’D K:“orhit”l &“,,“\, $ADH Vrn forward
Vreverw
KF”‘b. NAD
.‘;,“C. NADH $4” K’“‘b”“I K ‘WC,““< &‘I *All
values
constants for dehydrogenase 0.0818* 0.38 1 136 0.0669 0.229-F
5.84t 0.091$ O.SSOt: 0.238 I.1 I 13.1 0.002 14 are given as mM +
rat
liver
dehydrogenase
653
kinetics
50
sorbitol
io.0105 f 0.0390 f 14.2 +0.0075 * 0.0084 i_ 0.407 * 0.02 I kO.32 f 0.0328 +0.1x8 f 3.74 i 0.005 18
I SD unless otherwise
specified. t Given as pmolimin per mg of enzyme protein. : Expressed as mM*.
was observed for each of the substrates beginning at approximately the stated value and increased with increasing concentrations: sorbitol (49 mM); NAD+ (7 mM); D-fructose (250 mM) and NADH (0.4 mM).
0
2
4
I/Sorbitol,
6
mM_’
Product inhibition studies reciprocal plots arising from the product experiments in the forward direction (i.e. with fructose as the inhibitor and sorbitol and NAD+ as the variable substrates, respectively) showed competitive inhibition. The corresponding plots with sorbitol (Fig. 2A) and NAD+ as varying substrates against NADH as the inhibitor were also indicative of competitive inhibition. Secondary slope plots were linear, except for the two “concave upward” replots for the variable substrate, NAD+, against fructose and NADH as inhibitors. Product inhibition studies in the reverse (ketose reduction) direction, performed with D-fructose and NADH as the variable substrates with sorbitol and NAD+ as inhibitors, respectively, showed competitive inhibition. The secondary slope replot for the variable substrate NADH with NAD+ as the inhibitor was linear. The slope replot was “concave upward” for the variable substrate D-fructose with sorbitol as the inhibitor. Inhibition patterns for fructose as the variable substrate against NAD+ as the inhibitor (Fig. 2B) and NADH as variable substrate against sorbitol as inhibitor were non-competitive. All of the non-competitive slope and intercept replots were linear. A summary of these product inhibition patterns and kinetic constants is shown in Table 2. Additionally, all four p&duct inhibition studies in the forward direction were carried out at saturating concentrations of fixed substrate (> 100 K,). Under these conditions no inhibition should be observed for a rapid equilibrium random mechanism at subsaturating levels of product. With NADC as the variable substrate and NADH as the inhibitor (in the presence of saturating sorbitol) the inhibition pattern remained competitive. Competitive inhibition was also retained with sorbitol as the variable substrate and fructose as the inhibitor (in the presence of saturating NAD+). These results are indicative of abortive complex formation. Double inhibition
:
I 0
I
I
I
0.025
I/Fructose.
0.050
0.075
J 0.100
mM_’
Fig. 2A. Product inhibition double reciprocal plot with NADH as the inhibitor and sorbitol as the variable substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50mM HEPES, 0.10 ionic strength; 24 pg of enzyme; 2.0 mM NAD+ ; sorbitol (as indicated) and NADH [absent (o), 0.009 mM (0). 0.018 mM (A), 0.028 mM (0). 0.038 mM (m), 0.048 mM (A)]. (-0) designates slope replot. B. Product inhibition double reciprocal plot with NAD+ as the inhibitor and fructose as the variable substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50 mM HEPES, 0.10 ionic strength: 24pg of enzyme; 0.035 mM NADH; fructose (as indicated) and NAD+ [absent (o),0.5 mM (0, 1.0 mM @I), 1.5 mM (0). 2.0 mM (m), 2.5 mM (A)]. (t-0) and (A---A) refer to slope and intercept replots. respectively.
654
NANCY C. LEESING and EUGENE T. MCGUINNESS
Table 2. Summary of product inhibition patterns and kinetic constants for rat-liver sorbitol dehydrogenase Inhibition constants Substrate Inhibitor Forward direction NADH Fructose NADH Fructose Reverse direction NAD+
Sorbitol NAD+ Sorbitol
Inhibition
K,,, f ISE*
Var
Fixed
type
(n-M)
NAD+ NAD+ Sorbitol Sorbitol
Sorbitol Sorbitol NAD+ NAD+
Parabolic camp. Parabolic camp. Linear camp. Linear camp.
0.0041 I.4 0.014 5.8
+ + & k
0.00046 0.70 0.0005 0.26
NADH NADH Fructose Fructose
Fructose Fructose NADH NADH
Linear camp. Linear non-camp. Linear non-camp. Parabolic camp.
0.28 359 II 20
* * + k
0.022 337 7.7 1.6
0.00040 I.0 X IWh
23 * 1.3 0.67 & 0.043 428
*K ,s, = K,, for lmear replots. K,s2 is the second inhibition constant provided by the parabolic program when data were fit to the equation c = (V.A)/[K(l + I/KIsz + IZ.K,s2) + A].
DISCUSSION
Initial velocity patterns obtained with L-iditol dehydrogenase for sorbitol oxidation and fructose reduction are consistent with a sequential addition and release of reactants and rule out a substituted enzyme mechanism. The simplest sequential mechanism compatible with the product inhibition data for sorbitol oxidation is the rapid equilibrium random mechanism with no abortive ternary complexes. The validity of the rapid equilibrium assignment is supported by the value for k,,, [S sect’, estimated from the amount of pure enzyme using the fold purification for Step 5. Table I of (Leissing & McGuinness, 1978)] and the assumption of a single active site on the enzyme. Even allowing for three or four active sites on the enzyme. the value estimated for k,,, is still within the range of 30-50 sect 1 as proposed by Purich et u/. (1977) consistent with this assignment. Substrate activation by sorbitol is clearly indicated by the non-linear slope replot obtained from initial velocity studies. (The comparable effect arising from NAD+ is very slight or negligible.) We can estimate the extent of rate enhancement for each substrate from the ordinate intercepts (Z&U’,,) using the computer generated equation for each of the fitted curves. These values (V, = 0.28 pmol/min per mg for sorbitol and V, = 0.24 pmol/min per mg for NAD+ as the changing-fixed substrates, respectively) when compared with the Cleland computer program generated value for V, (0.23 pmol/min per mg) based on linear secondary plots. indicate the activation effect is about 22”; for sorbitol and Cr40/ for NAD’. At low levels of sorbitol this rate enhancement effect would provide an alternate means for the metabolism of polyol. Although the data of Christensen et a/. (1975) were fit to a linear equation, a sorbitol activation effect is also indicated by the data points in the slopes replot [Fig. I of Christensen et u[. (1975)]. The abortive ternary complexes, E-sorbitolNADH and E-fructose-NAD, are not kinetically significant in the direction of sorbitol oxidation, since all four of the product inhibition patterns are competitive. Evidence for the kinetic identity of an abortive E-sorbitollNADH complex with the sheep liver enzyme is based on the non-competitive inhibition
K,, & ISE (mM)
K IS2* (mM’)
competitive
computer
observed when sorbitol is the variable substrate with NADH as the inhibitor [Fig. 3 of (Christensen ct 01.. 1975)]. However. the formation of both abortive complexes with the rat liver enzyme can be inferred from the parabolic slope replots observed with NAD’ as the variable substrate with fructose (Table 2) and NADH as inhibitors, Their existence is confirmed when the product inhibition studies with fructose and NADH are carried out in the presence of saturating concentrations of NAD+ and sorbitol as fixed substrates. In two respects the sheep and rat liver enzymes are similar in the forward direction. First, the relationship between the kinetic constants, Ki;K, = K,,.K,, is satisfied for each and the values (rat: 0.091 mM2; sheep 0.15 mM2) are in good agreement with each other. Second. estimates of k,,, (rat: 8 sec..‘; sheep: 5 sect ‘) are low enough to be consistent with the rapid equilibrium mechanism which assumes that the catalytic step is slow compared with ligand binding and dissociation steps. Frieden (1976) has pointed out that a rapid equilibrium ordered mechanism with abortive EB and EP complexes will show the same product inhibition patterns as a random addition of substrates. but with ternary complex conversion as a rate limiting step. While this possibility cannot be rigorously excluded for cases where k,,,is less than 10 set- ’ without isotope partitioning studies (Purich er al., 1977). the presence of an abortive E-sorbitol complex is contraindicated by the evidence of sorbitol activation for both the rat and sheep liver enzymes. In addition, the rapid equilibrium assumption does not appear to hold for the reverse reaction. This alternate proposal, it should be noted, does not preclude the formation of abortive EAP or EQB complexes. The product inhibition patterns found for the enzyme in the reverse direction (Table 2) initially indicate a rapid equilibrium random mechanism with two abortive ternary complexes, or a Theorell-Chance mechanism without abortive complexes, However, neither of these assignments is in accord with other criteria derived from the data. The value of k,,, for fructose reduction is estimated to be 190 sect 1 [from activity of Step 5, Table I of Leissing & McGuinness (1978)] for the rat liver enzyme. Purich et
655
Sorbitol dehydrogenase kinetics have proposed that the rapid equilibrium assumption does not hold when k,,, exceeds 3c-50 set-‘. The Theorell-Chance mechanism does not provide a reasonable explanation of the data, since the relationship, I - ((V,.K,)/( V;Kia)) = 0, is not satisfied (Cleland, 1963) for NADH or fructose. Additionally, a crossover point analysis of the data (Janson & Cleland. 1974) does not support a TheorellLChance mechanism. Whereas the presence of the abortive ternary complexes in the forward direction is not kinetically significant and does not impede the reaction, these complexes become kinetically important in the reverse (ketose reduction) direction. If we designate NAD+. sorbitol, D-fructose and NADH as A, B, P, and Q, respectively, the complexes and their dissociation constants for NAD’ and sorbitol can be written: EAP = EA + A (K,,) and EBQ = EQ + B (K,,). Estimates of the values of these constants + 1 SD are generated using Eqns 2 and 3.
(B) KlH -1
1
(2)
(3) By this approach the rapid equilibrium random mechanism with the abortive ternary complexes EAP and EBQ (Eqns 2 and 3) fits the data with K,, = 0.308 & 0.063 mM and K,, = 16.2 + 1.6 mM. Since these dissociations are part of closed cycles in equilibria (Stayton & Fromm (1979), K,,, EB + Q = EBQ = EQ + B. K,,
(4)
K,,
(5)
and EA + P = EAP = EP + A, K,,
the dissociation constants K,, and K,, can be estimated from the values of K,,4 and K,, and the relations: Ki,.K,,
= K,,,K,,
(6)
Kio.K,,
= K,Q.Ki,
(7)
and
These values are: K,, = 17mM; K,, = 0.031 mM. Although K,, and K,, are the same (16 and 17 mM). the inhibition patterns in the forward direction remain competitive because the effect of the [I + (1)/K,,] component on the intercept term is not large enough to be observed. Specifically, the ratio CK,(I + (WK,Jl/G is -3 for K,, and K,, and -7-9 for K,, and K,,. In comparing the rat and sheep liver enzymes in the reverse (fructose reduction) direction, the calculated k,,, value of 190 set -’ for the rat liver enzyme is higher than the value of 74 set- ’ for the sheep liver enzyme. However, both exceed the 30-50 set-’ range
proposed by Purich et al. (1977) for the rapid equilibrium mechanism assumption. For both enzymes the relationship K,. K, = K,,.K, is satisfied, and if we allow for the relatively large error in KpADH(Table 1) and assume the value of K,NADH= 11.I PM for the sheep liver enzyme [Table 3 of Christensen et al. (1975)], the values are in agreement for the two enzymes. A proposed mechanism A composite picture of the sequence of kinetic events, including the role of the dead-end complexes, emerges from a consideration of both forward and reverse reactions. When rapid equilibrium conditions prevail, all steps involving binding and releasing in the enzyme complex formation equilibrate rapidly relative to the transformation(s) of the central complex(s). Thus, in the presence of fructose as inhibitor, the E-fructose-NAD+ abortive complex rapidly forms and dissociates fructose and E-NAD+. In turn the rapid equilibration of E-NAD+ provides, at low levels of sorbitol, sufficient E and E-NAD+ for the productive ternary complex(s) E-sorbitolLNAD’ to form. The relative magnitudes of the dissociation constants KIP, K, K, and Ki, are compatible with this interpretation and in effect, the presence of the E-fructoseeNAD+ complex is not manifest under these conditions. From our data. this complex is “seen” when the competitive inhibition of fructose and sorbitol is studied at saturating levels of NAD+. Theory dictates that in the absence of the abortive complex the (competitive) inhibition should not be observed since the level of E becomes insignificant at saturating NAD+. However, the inhibition remains competitive since sorbitol and fructose are now competing for the E-NAD’ complex. Again, the values of K,, and K, support this view. An entirely analogous picture can be presented for the E-sorbitol-NADH complex. Thus, at low levels of sorbitol and cofactor, polyol oxidation is not significantly affected by the presence of either of these abortive complexes. In contrast. the abortive complexes are kinetically evident in the direction of fructose reduction. This situation arises since central complex conversion is not the rate limiting step (k,,, > 190 secC’) and, in the presence of sorbitol or NAD+ as inhibitors, one or more of the binding/release steps involving abortive binary or ternary complexes is slower than the chemical step. Consequently each reactant (NADH and fructose) is faced with a deficit of an enzyme specie (E, E-NADH, E-fructose) requisite for ketose reduction. A summary depiction of these events is shown in Scheme 1. While the initial velocity equation for a A I
KIb
I
6
B I
r’
I
K,
A
EBQ b
C’ &
II I
K9
0
A
EAP
Kip
P
NANCY C. LEISSING and EUGENE T. MCGUINNESS
656
random mechanism predicts nonlinearity in double reciprocal plots, computer modeling studies indicate that this effect may not always be discernible (Fromm, 1979). We note, however, as Fromm (1979) has pointed out, that very few random bi bi mechanisms are truly rapid equilibrium random in both directions although this condition will be approximated in the “slow direction”.
SUMMARY
Steady state initial velocity and product inhibition experiments were carried out in the direction of polyol oxidation and fructose reduction with the enzyme sorbitol dehydrogenase (L-iditol: NAD-oxidoreductase, EC 1.1.1.14),previously purified from rat liver. Data analyses indicate the enzyme follows a rapid equilibrium random mechanism in the direction of sorbitol oxidation and a random mechanism in the direction of fructose reduction. k,,, values for polyol oxidation and fructose reduction are estimated to be - 8 set- ’ and - 190 set- I, respectively. Substrate activation by sorbitol is suggested at low levels of this reactant. The presence of the abortive ternary complexes E-NADH-Sorbitol and E-Fructose-NAD+ is demonstrated, but they are not kinetically significant in the direction of polyol oxidation. These data are compared with results reported by Christensen et al. (1975) for sheep-liver enzyme. The role proposed here for the abortive ternary complexes in the rat-liver enzyme may have clinical implications relative to the behavior of sorbitol dehydrogenase in those tissues in which the polyol concentration is sufficiently high to be of osmotic importance.
Acl\,loM/edUrrttr/lrsThis by Travenol Laboratories.
work
was supported,
in part,
REFERENCES BLAKLEY R. L. (1951) The
metabolism and antiketogenic effects of sorbitol. Sorbitol dehydrogenase. Biocl~em. J. 49. 257-271. BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. A&JY. Biochrm. 72. 248-254. CHRIST~~VSI:~U.. TUSTHEN E. & AN~ERSEN B. (1975) Initial velocity and product inhibition studies on L-iditol: NAD oxidoreductase. Actu chrm. swnd. 29B, 8lLX7. CLELANI) W. W. (1963) The kinetics of enzyme-catalyzed
reactions with two or more substrates or products. II. Inhibition: Nomenclature and theory. Biochim. hiophys. Actu 67, 173-l 87. CLELAND W. W. (1967) The statistical analysis of enzyme kinetic data. Ah. EIIZVPI. 29, I -32. CLELAND W. W. (1979) Statistical analysis of enzyme kinetic data. Mrrh. E~IzY~)~. 63. IO3 138. FINI: I. H. & COSTF:I.LO L. A. (1963) The use of starch electrophoresis in dehydrogenase studies. ,2lrrh. ~+ZZJ,,II. 6, 95% 962. FKIEDE~ C. (1976) On the kinetic distinction of ordered and random bioreactant enzyme systems. B~oc~/Ic,~II. h~ophps. Rrs. C’o/mur~. 68. 9 I449 17. FROMM H. J. (1975) Inititrl Rote EJIZ~~J~J Kiwtic.\. SpringerVerlag. Heidelberg. FROMM H. J. (1979) Use of competitive inhibitors to study substrate binding order. ,%frt/~. Ej~:),,n. 63. 467-4X6. FROMM H. J. & Nr~sou D. R. (1962) Ribitol dehydrogenase. III. Kinetic studies with product inhibition. J. hid. Chrm. 237. 2 15-220. HOCKING; R. R. & LESLIE R. N. (1967) Selection of the best subset in regression analysis. 7’rc,/?rlor,l~lrit,.s 9. 53 I-540. JANSON C. A. & CLLLANI) W. W. (1974) The kinetic mechanism of glycerokinase. J. hiol. Chr,r~~.249. 2562 7566. JEDZINIAK J. A.. CHYLA(.K L. T.. CH~N(; H-M.. GII.I.IS M. K.. KALUSTIAU A. A. & TLN(; W. H. (19X1) The sorbitol pathway in the human lens: Aldose reductase and polyol dehydrogenase. I~IYY~. Op/~r/ltr/. l’is. %i. 20. 3 144326. JIZFFERYJ.. CUMMLYS L.. CARLQLIIST M. & JORI*;VALL H. (1981) Properties of sorbitol dehydrogenasc and characterization of a reactive cysteine residue reveal uncxpetted similarities to alcohol dchydrogcnase. Eur. J. Bio~/IUJJI.120, 219-234. JORNVALL H.. P~RSSO~ M. & JI:FFI:RY J. (19x1) Alcohol and polyol dehydrogenases arc both divided into two protein types, and structural propertics cross-related the diffcrent enzyme activities with each type. Proc. ~I(/~JI.Ac~trd. SC;.. C’.S.A. 78, 4226 4130. LEISSINC; N. & MCGUII\;N~SS E. T. (197X) Rapid affinity purification and properties of rat liver sorbitol dchydrogenase. Biochim. hioph_v.s. Acfrr 524. 254 26 I. PURICH D. L.. ALLISOI\I R. D. & TODHLNT~R J. A. (1977) On the rapid equilibrium assumption and the problem of distinguishing certain ordered and random kinetic mechanisms. Eioc/lc,n~. hioph~s. Rcs. Comnm. 77, 753 759. STAYTON M. M. & FROMM H. J. (1979) Purifcation. properties. and kinetics of I,-ribulokinase from Aerobatter aerogenes. J. bid. C/~em. 254. 3765-377 I, TSAI C. S. (1982) Multifunctionality of liver alcohol dehydrogenase: Kinetic and mechanistic studies of csterase reaction. Arch Biochrrn. Biop/~vs. 213. 635-642. UE~‘C; S. T-H. & MCGLIIKNLSS ‘E. T. 11977) I,-Mannitol dehydrogenase from Absidfrr q/~mcu. Steady-state kinetic properties and the inhibitory role of mannitol I-phosphate. Biochrr~listry 16, 107- I I I. WILKINSON G. N. (1961) Statistical estimations in enzyme kinetics. Bioc~hrrn. J. 80. 324333.
KINETIC
Department
0020-711X.83~050651-06$03.00;‘0 Copyright 0 1983 Pergamon Press Ltd
ANALYSIS OF RAT LIVER DEHYDROGENASE
SORBITOL
NANCY C. LEISSING* and EUGENE T. MCGUINNESS~ of Chemistry, Seton Hall University. South Orange, NJ 07079. U.S.A. (Rrceired
2 Augusf
1982)
Abstract-l, A steady state kinetic investigation was performed on an improved preparation of rat-liver sorbitol dehydrogenase (L-iditol: NAD-oxidoreductase, EC 1.1.1.14). 2. Data analyses indicate the enzyme follows a rapid equilibrium random mechanism in the direction of sorbitol oxidation and a random mechanism in the direction of fructose reduction. 3. Kinetic constants were: KEAD 0.082 mM; Kzrbi’“’ 0.38 mM; KiADH 67 pm; .k”““” 136 mM. 4. Evidence is adduced to indicate the more rapid reverse (fructose reduction) reaction is susceptible to metabolic control by formation of abortive enzymeefructose-NAD and enzyme-NADH-sorbitol com-
plexes.
INTRODUCTION
Comparative amino acid sequence studies demonstrate that the enzymes, sheep liver sorbitol dehydrogenase (SDH), horse liver alcohol dehydrogenase (LADH) and Drosophilia alcohol dehydrogenase (DADH) provide examples of dehydrogenases that have different substrates (ethanol and sorbitol) but are structurally related (LADH and SDH), and of dehydrogenases that utilize the same substrate (ethanol) though they (DADH and LADH) lack close structural relationships (Jeffery et al., 1981; Jornvall ef al., 1981). Similarly, Klebsiella ribitol dehydrogenase (RDH) is revealed to have a closer structural homology to DADH than to SDH. While rate studies on ADH continue to generate useful insight about its function (e.g. Tsai, 1982) comparable information on the polyol dehydrogenases is sparse or unavailable. Ribitol dehydrogenase from Aerobacter aerogenes, for example, was found to follow an ordered bi bi mechanism involving ribitol and D-ribulose as the inside reactant pair with the formation of two abortive ternary complexes (Fromm & Nelson, 1962). D-Mannitol dehydrogenase from Absi&a glauca was shown to follow an ordered mechanism which is uniquely susceptible to metabolic control by mannitol l-phosphate acting as a dead-end inhibitor (Ueng & McGuinness, 1977). Sorbitol dehydrogenase from sheep liver is reported to obey a rapid equilibrium random mechanism in both directions that includes the formation of an abortive sorbitolenzyme-NADH complex (Christensen et al., 197.5). Correlative studies of structure-function relationships the alcohol-polyol dehydrogenases will among depend on a better understanding of the kinetic behavior of enzymes in the latter category.
The present report deals with a steady state kinetic investigation of an improved preparation (Leissing & McGuinness, 1978) of rat-liver sorbitol dehydrogenase, first partially purified from this tissue by Blakley (1951). Data analysis reveals the presence of kinetically significant abortive ternary complexes in the direction of fructose reduction. It remains to be established whether this characteristic is a property unique to the liver enzyme or is also an attribute, and of metabolic significance, to lens sorbitol dehydrogenase (Jedziniak et al., 1981).
MATERIALS AND METHODS Sorbitol and fructose were obtained from Pfanstiehl Laboratories. Proteins and nucleotide cofactors were purchased from Sigma Chemical Co. Sephacryl S-200 was obtained from Pharmacia. Buffers, including Tris-HCI and HEPES (N-2-hydroxyethyl piperazine-N’-2-ethane sulfonic acid) were procured from Calbiochem-Behring Corp. Electrophoresis support media was purchased from Beckman Instruments, Inc. All other chemicals were reagent grade or of superior quality. Enzpe
preparation
Sorbitol dehydrogenase from rat liver was purified up to and including Step 3 as discussed elsewhere (Leissing & McGuinness, 1978). Purification Step 4 was performed using a Sephacryl G-200 column (2.6 x 25cm) previously equilibrated with 10mM phosphate buffer, pH 7.5, containing 1 mM DDT (dithiothreitol). Elution was carried out in an upward direction using a flow rate of about 60 ml/hr. Active fractions from several runs were pooled, divided into aliquots and stored at - 2o’C until used. The partially purified enzyme preparation was stable in this form for at least 6 months. This preparation was judged to be enzymically homogeneous based on the consistent relative rates of oxidation by various polyols for Steps 3-5 (Leissing & McGuinness. 1978). Protein and unz~me vmsurements
* Present address: Department of Safety Assessment, Travenol Laboratories, Morton Grove, IL 60053, U.S.A. t To whom correspondence and reprint requests should be addressed.
Protein measurement was carried out after precipitation with tricholoacetic acid using a Coomassie blue protein assay as described by Bradford (1976). Bovine serum 651
NANCY C. LEISSING and EUGENE T. MCGLJINNESS
652
albumin was used as the standard. Estimate of the enzyme content was performed with the Beckman Microzone Cell. Duostat and R-l 12/l 15 scanning densitometer using pH 8.6 barbital buffer and ponceau S dye with visualization of the single activity band by staining with sorbitol, NAD+, phcnazine methosulfate and nitroblue tetrazolium, according to Fine & Costello (1963).
Sorbitol dehydrogenase kinetic studies were performed using a Gilford 3500 Automatic Analyzer with General Kinetic Card No. 5. The rate of change of absorbance at 340nm was followed at 25 C. For the identification of enzyme location from gel chromatography tubes the assay system contained 1.3 mM NAD’, 7.3 mM sorbitol. 50 mM Tris+HCl buffer (pH 9.6) and 0.1 ml enzyme preparation in a total volume of 1.35 ml. For initial velocity and product inhibition studies in both the forward and reverse directions, the assay system contained 50 mM HEPES Butler (pH 7.5), ionic strength 0.1 with the NaCI. and up to 0.1 ml of the pooled Step 4 enzyme preparation in a total volume of 1.35 ml. All reaction progress curves were linear for several minutes for the concentrations of substrate used. Blank reactions containing all components except alternate substrate were used as controls in all studies. Each experimental run was carried out in duplicate. Data from preliminary initial velocity studies were evaluated by computer fitting to the equation
C’IIAI
” = K,+CAI
SO
0
r
12l
I
I
I
I/Sorbitol,
mM-’
(1)
using a weighted least-squares numeric coefficient calculation (Wilkinson. 1961). At the ligand concentrations used the errors were found to conform to a normal distribution with approximate equal variances. Subsequently, all initial-velocity data were analyzed using the appropriate rate equations and computer programs described by Cleland (1967. 1979). In addition. replots of slopes and intercepts were examined for linearity using the Statistical Analysis System (SAS) software package with the General Linear Model (GLM) procedure. When the u~.Y’ term (.v = a,, + U,Y + as_?) was significant at the P < 0.05 level. the replot was judged to be nonlinear. Product inhibition data were also evaluated using the model-testing (as opposed to model-fitting) approach, as discussed by Fromm (197.5) and Hocking & Leslie (1967), to verify the nature of each inhibition pattern.
RESULTS
I/NADH, mM-’
I
I
I
I
0.02
0.04
0.06
0.06
I /Fructose, These were carried out using NAD+ and sorbitol
I 6
4
2
in the forward direction (Fig. 1A) as the variable
substrates against sorbitol and NAD+ as the changing-fixed substrates, respectively. In the reverse direction, D-fructose and NADH were the variable substrates. respectively, with NADH and D-fructose (Fig. 1B) as the changing-fixed substrates. All double reciprocal primary plots were intersecting. All slope and intercept replots were linear as determined by the procedure described in Materials and Methods, except for the “concave downward” slope replots generated by the reactions with NAD+ and sorbitol (Fig. IA) as variable substrates. A summary of the computer calculated kinetic parameters from these initial velocity studies is shown in Table 1. Inhibition
I 0. IO
mM-’
Fig. 1A. Initial velocity double reciprocal plot with sorbitol as the variable and NAD+ as the changing fixed substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50mM HEPES, 0.10 ionic strength; 29 pg of enzyme; sorbitol (as shown) and NAD+ [0.05 mM (O), 0.08 mM (0). 0.10 mM (a), 0.14 mM (o), 0.25 mM (m), l.OOmM (A). (00) and (A--A) refer to slope and intercept replots. respectively. B. Initial velocity double reciprocal plot with fructose as the variable and NADH as the changing fixed substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50mM HEPES, 0.10 ionic strength; 29jig of enzyme; fructose (as shown) and NADH CO.022 mM (0). 0.027 mM (O), 0.035 mM (A). 0.048 mM (0). 0.077 mM (w)], (-0) and (A---A) refer to slope and intercept replots, respectively.
Sorbitol Table
1.
Kinetic
KN’D K:“orhit”l &“,,“\, $ADH Vrn forward
Vreverw
KF”‘b. NAD
.‘;,“C. NADH $4” K’“‘b”“I K ‘WC,““< &‘I *All
values
constants for dehydrogenase 0.0818* 0.38 1 136 0.0669 0.229-F
5.84t 0.091$ O.SSOt: 0.238 I.1 I 13.1 0.002 14 are given as mM +
rat
liver
dehydrogenase
653
kinetics
50
sorbitol
io.0105 f 0.0390 f 14.2 +0.0075 * 0.0084 i_ 0.407 * 0.02 I kO.32 f 0.0328 +0.1x8 f 3.74 i 0.005 18
I SD unless otherwise
specified. t Given as pmolimin per mg of enzyme protein. : Expressed as mM*.
was observed for each of the substrates beginning at approximately the stated value and increased with increasing concentrations: sorbitol (49 mM); NAD+ (7 mM); D-fructose (250 mM) and NADH (0.4 mM).
0
2
4
I/Sorbitol,
6
mM_’
Product inhibition studies reciprocal plots arising from the product experiments in the forward direction (i.e. with fructose as the inhibitor and sorbitol and NAD+ as the variable substrates, respectively) showed competitive inhibition. The corresponding plots with sorbitol (Fig. 2A) and NAD+ as varying substrates against NADH as the inhibitor were also indicative of competitive inhibition. Secondary slope plots were linear, except for the two “concave upward” replots for the variable substrate, NAD+, against fructose and NADH as inhibitors. Product inhibition studies in the reverse (ketose reduction) direction, performed with D-fructose and NADH as the variable substrates with sorbitol and NAD+ as inhibitors, respectively, showed competitive inhibition. The secondary slope replot for the variable substrate NADH with NAD+ as the inhibitor was linear. The slope replot was “concave upward” for the variable substrate D-fructose with sorbitol as the inhibitor. Inhibition patterns for fructose as the variable substrate against NAD+ as the inhibitor (Fig. 2B) and NADH as variable substrate against sorbitol as inhibitor were non-competitive. All of the non-competitive slope and intercept replots were linear. A summary of these product inhibition patterns and kinetic constants is shown in Table 2. Additionally, all four p&duct inhibition studies in the forward direction were carried out at saturating concentrations of fixed substrate (> 100 K,). Under these conditions no inhibition should be observed for a rapid equilibrium random mechanism at subsaturating levels of product. With NADC as the variable substrate and NADH as the inhibitor (in the presence of saturating sorbitol) the inhibition pattern remained competitive. Competitive inhibition was also retained with sorbitol as the variable substrate and fructose as the inhibitor (in the presence of saturating NAD+). These results are indicative of abortive complex formation. Double inhibition
:
I 0
I
I
I
0.025
I/Fructose.
0.050
0.075
J 0.100
mM_’
Fig. 2A. Product inhibition double reciprocal plot with NADH as the inhibitor and sorbitol as the variable substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50mM HEPES, 0.10 ionic strength; 24 pg of enzyme; 2.0 mM NAD+ ; sorbitol (as indicated) and NADH [absent (o), 0.009 mM (0). 0.018 mM (A), 0.028 mM (0). 0.038 mM (m), 0.048 mM (A)]. (-0) designates slope replot. B. Product inhibition double reciprocal plot with NAD+ as the inhibitor and fructose as the variable substrate. Initial velocities are expressed as pmol/min per mg. Reaction mixture contained: 50 mM HEPES, 0.10 ionic strength: 24pg of enzyme; 0.035 mM NADH; fructose (as indicated) and NAD+ [absent (o),0.5 mM (0, 1.0 mM @I), 1.5 mM (0). 2.0 mM (m), 2.5 mM (A)]. (t-0) and (A---A) refer to slope and intercept replots. respectively.
654
NANCY C. LEESING and EUGENE T. MCGUINNESS
Table 2. Summary of product inhibition patterns and kinetic constants for rat-liver sorbitol dehydrogenase Inhibition constants Substrate Inhibitor Forward direction NADH Fructose NADH Fructose Reverse direction NAD+
Sorbitol NAD+ Sorbitol
Inhibition
K,,, f ISE*
Var
Fixed
type
(n-M)
NAD+ NAD+ Sorbitol Sorbitol
Sorbitol Sorbitol NAD+ NAD+
Parabolic camp. Parabolic camp. Linear camp. Linear camp.
0.0041 I.4 0.014 5.8
+ + & k
0.00046 0.70 0.0005 0.26
NADH NADH Fructose Fructose
Fructose Fructose NADH NADH
Linear camp. Linear non-camp. Linear non-camp. Parabolic camp.
0.28 359 II 20
* * + k
0.022 337 7.7 1.6
0.00040 I.0 X IWh
23 * 1.3 0.67 & 0.043 428
*K ,s, = K,, for lmear replots. K,s2 is the second inhibition constant provided by the parabolic program when data were fit to the equation c = (V.A)/[K(l + I/KIsz + IZ.K,s2) + A].
DISCUSSION
Initial velocity patterns obtained with L-iditol dehydrogenase for sorbitol oxidation and fructose reduction are consistent with a sequential addition and release of reactants and rule out a substituted enzyme mechanism. The simplest sequential mechanism compatible with the product inhibition data for sorbitol oxidation is the rapid equilibrium random mechanism with no abortive ternary complexes. The validity of the rapid equilibrium assignment is supported by the value for k,,, [S sect’, estimated from the amount of pure enzyme using the fold purification for Step 5. Table I of (Leissing & McGuinness, 1978)] and the assumption of a single active site on the enzyme. Even allowing for three or four active sites on the enzyme. the value estimated for k,,, is still within the range of 30-50 sect 1 as proposed by Purich et u/. (1977) consistent with this assignment. Substrate activation by sorbitol is clearly indicated by the non-linear slope replot obtained from initial velocity studies. (The comparable effect arising from NAD+ is very slight or negligible.) We can estimate the extent of rate enhancement for each substrate from the ordinate intercepts (Z&U’,,) using the computer generated equation for each of the fitted curves. These values (V, = 0.28 pmol/min per mg for sorbitol and V, = 0.24 pmol/min per mg for NAD+ as the changing-fixed substrates, respectively) when compared with the Cleland computer program generated value for V, (0.23 pmol/min per mg) based on linear secondary plots. indicate the activation effect is about 22”; for sorbitol and Cr40/ for NAD’. At low levels of sorbitol this rate enhancement effect would provide an alternate means for the metabolism of polyol. Although the data of Christensen et a/. (1975) were fit to a linear equation, a sorbitol activation effect is also indicated by the data points in the slopes replot [Fig. I of Christensen et u[. (1975)]. The abortive ternary complexes, E-sorbitolNADH and E-fructose-NAD, are not kinetically significant in the direction of sorbitol oxidation, since all four of the product inhibition patterns are competitive. Evidence for the kinetic identity of an abortive E-sorbitollNADH complex with the sheep liver enzyme is based on the non-competitive inhibition
K,, & ISE (mM)
K IS2* (mM’)
competitive
computer
observed when sorbitol is the variable substrate with NADH as the inhibitor [Fig. 3 of (Christensen ct 01.. 1975)]. However. the formation of both abortive complexes with the rat liver enzyme can be inferred from the parabolic slope replots observed with NAD’ as the variable substrate with fructose (Table 2) and NADH as inhibitors, Their existence is confirmed when the product inhibition studies with fructose and NADH are carried out in the presence of saturating concentrations of NAD+ and sorbitol as fixed substrates. In two respects the sheep and rat liver enzymes are similar in the forward direction. First, the relationship between the kinetic constants, Ki;K, = K,,.K,, is satisfied for each and the values (rat: 0.091 mM2; sheep 0.15 mM2) are in good agreement with each other. Second. estimates of k,,, (rat: 8 sec..‘; sheep: 5 sect ‘) are low enough to be consistent with the rapid equilibrium mechanism which assumes that the catalytic step is slow compared with ligand binding and dissociation steps. Frieden (1976) has pointed out that a rapid equilibrium ordered mechanism with abortive EB and EP complexes will show the same product inhibition patterns as a random addition of substrates. but with ternary complex conversion as a rate limiting step. While this possibility cannot be rigorously excluded for cases where k,,,is less than 10 set- ’ without isotope partitioning studies (Purich er al., 1977). the presence of an abortive E-sorbitol complex is contraindicated by the evidence of sorbitol activation for both the rat and sheep liver enzymes. In addition, the rapid equilibrium assumption does not appear to hold for the reverse reaction. This alternate proposal, it should be noted, does not preclude the formation of abortive EAP or EQB complexes. The product inhibition patterns found for the enzyme in the reverse direction (Table 2) initially indicate a rapid equilibrium random mechanism with two abortive ternary complexes, or a Theorell-Chance mechanism without abortive complexes, However, neither of these assignments is in accord with other criteria derived from the data. The value of k,,, for fructose reduction is estimated to be 190 sect 1 [from activity of Step 5, Table I of Leissing & McGuinness (1978)] for the rat liver enzyme. Purich et
655
Sorbitol dehydrogenase kinetics have proposed that the rapid equilibrium assumption does not hold when k,,, exceeds 3c-50 set-‘. The Theorell-Chance mechanism does not provide a reasonable explanation of the data, since the relationship, I - ((V,.K,)/( V;Kia)) = 0, is not satisfied (Cleland, 1963) for NADH or fructose. Additionally, a crossover point analysis of the data (Janson & Cleland. 1974) does not support a TheorellLChance mechanism. Whereas the presence of the abortive ternary complexes in the forward direction is not kinetically significant and does not impede the reaction, these complexes become kinetically important in the reverse (ketose reduction) direction. If we designate NAD+. sorbitol, D-fructose and NADH as A, B, P, and Q, respectively, the complexes and their dissociation constants for NAD’ and sorbitol can be written: EAP = EA + A (K,,) and EBQ = EQ + B (K,,). Estimates of the values of these constants + 1 SD are generated using Eqns 2 and 3.
(B) KlH -1
1
(2)
(3) By this approach the rapid equilibrium random mechanism with the abortive ternary complexes EAP and EBQ (Eqns 2 and 3) fits the data with K,, = 0.308 & 0.063 mM and K,, = 16.2 + 1.6 mM. Since these dissociations are part of closed cycles in equilibria (Stayton & Fromm (1979), K,,, EB + Q = EBQ = EQ + B. K,,
(4)
K,,
(5)
and EA + P = EAP = EP + A, K,,
the dissociation constants K,, and K,, can be estimated from the values of K,,4 and K,, and the relations: Ki,.K,,
= K,,,K,,
(6)
Kio.K,,
= K,Q.Ki,
(7)
and
These values are: K,, = 17mM; K,, = 0.031 mM. Although K,, and K,, are the same (16 and 17 mM). the inhibition patterns in the forward direction remain competitive because the effect of the [I + (1)/K,,] component on the intercept term is not large enough to be observed. Specifically, the ratio CK,(I + (WK,Jl/G is -3 for K,, and K,, and -7-9 for K,, and K,,. In comparing the rat and sheep liver enzymes in the reverse (fructose reduction) direction, the calculated k,,, value of 190 set -’ for the rat liver enzyme is higher than the value of 74 set- ’ for the sheep liver enzyme. However, both exceed the 30-50 set-’ range
proposed by Purich et al. (1977) for the rapid equilibrium mechanism assumption. For both enzymes the relationship K,. K, = K,,.K, is satisfied, and if we allow for the relatively large error in KpADH(Table 1) and assume the value of K,NADH= 11.I PM for the sheep liver enzyme [Table 3 of Christensen et al. (1975)], the values are in agreement for the two enzymes. A proposed mechanism A composite picture of the sequence of kinetic events, including the role of the dead-end complexes, emerges from a consideration of both forward and reverse reactions. When rapid equilibrium conditions prevail, all steps involving binding and releasing in the enzyme complex formation equilibrate rapidly relative to the transformation(s) of the central complex(s). Thus, in the presence of fructose as inhibitor, the E-fructose-NAD+ abortive complex rapidly forms and dissociates fructose and E-NAD+. In turn the rapid equilibration of E-NAD+ provides, at low levels of sorbitol, sufficient E and E-NAD+ for the productive ternary complex(s) E-sorbitolLNAD’ to form. The relative magnitudes of the dissociation constants KIP, K, K, and Ki, are compatible with this interpretation and in effect, the presence of the E-fructoseeNAD+ complex is not manifest under these conditions. From our data. this complex is “seen” when the competitive inhibition of fructose and sorbitol is studied at saturating levels of NAD+. Theory dictates that in the absence of the abortive complex the (competitive) inhibition should not be observed since the level of E becomes insignificant at saturating NAD+. However, the inhibition remains competitive since sorbitol and fructose are now competing for the E-NAD’ complex. Again, the values of K,, and K, support this view. An entirely analogous picture can be presented for the E-sorbitol-NADH complex. Thus, at low levels of sorbitol and cofactor, polyol oxidation is not significantly affected by the presence of either of these abortive complexes. In contrast. the abortive complexes are kinetically evident in the direction of fructose reduction. This situation arises since central complex conversion is not the rate limiting step (k,,, > 190 secC’) and, in the presence of sorbitol or NAD+ as inhibitors, one or more of the binding/release steps involving abortive binary or ternary complexes is slower than the chemical step. Consequently each reactant (NADH and fructose) is faced with a deficit of an enzyme specie (E, E-NADH, E-fructose) requisite for ketose reduction. A summary depiction of these events is shown in Scheme 1. While the initial velocity equation for a A I
KIb
I
6
B I
r’
I
K,
A
EBQ b
C’ &
II I
K9
0
A
EAP
Kip
P
NANCY C. LEISSING and EUGENE T. MCGUINNESS
656
random mechanism predicts nonlinearity in double reciprocal plots, computer modeling studies indicate that this effect may not always be discernible (Fromm, 1979). We note, however, as Fromm (1979) has pointed out, that very few random bi bi mechanisms are truly rapid equilibrium random in both directions although this condition will be approximated in the “slow direction”.
SUMMARY
Steady state initial velocity and product inhibition experiments were carried out in the direction of polyol oxidation and fructose reduction with the enzyme sorbitol dehydrogenase (L-iditol: NAD-oxidoreductase, EC 1.1.1.14),previously purified from rat liver. Data analyses indicate the enzyme follows a rapid equilibrium random mechanism in the direction of sorbitol oxidation and a random mechanism in the direction of fructose reduction. k,,, values for polyol oxidation and fructose reduction are estimated to be - 8 set- ’ and - 190 set- I, respectively. Substrate activation by sorbitol is suggested at low levels of this reactant. The presence of the abortive ternary complexes E-NADH-Sorbitol and E-Fructose-NAD+ is demonstrated, but they are not kinetically significant in the direction of polyol oxidation. These data are compared with results reported by Christensen et al. (1975) for sheep-liver enzyme. The role proposed here for the abortive ternary complexes in the rat-liver enzyme may have clinical implications relative to the behavior of sorbitol dehydrogenase in those tissues in which the polyol concentration is sufficiently high to be of osmotic importance.
Acl\,loM/edUrrttr/lrsThis by Travenol Laboratories.
work
was supported,
in part,
REFERENCES BLAKLEY R. L. (1951) The
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reactions with two or more substrates or products. II. Inhibition: Nomenclature and theory. Biochim. hiophys. Actu 67, 173-l 87. CLELAND W. W. (1967) The statistical analysis of enzyme kinetic data. Ah. EIIZVPI. 29, I -32. CLELAND W. W. (1979) Statistical analysis of enzyme kinetic data. Mrrh. E~IzY~)~. 63. IO3 138. FINI: I. H. & COSTF:I.LO L. A. (1963) The use of starch electrophoresis in dehydrogenase studies. ,2lrrh. ~+ZZJ,,II. 6, 95% 962. FKIEDE~ C. (1976) On the kinetic distinction of ordered and random bioreactant enzyme systems. B~oc~/Ic,~II. h~ophps. Rrs. C’o/mur~. 68. 9 I449 17. FROMM H. J. (1975) Inititrl Rote EJIZ~~J~J Kiwtic.\. SpringerVerlag. Heidelberg. FROMM H. J. (1979) Use of competitive inhibitors to study substrate binding order. ,%frt/~. Ej~:),,n. 63. 467-4X6. FROMM H. J. & Nr~sou D. R. (1962) Ribitol dehydrogenase. III. Kinetic studies with product inhibition. J. hid. Chrm. 237. 2 15-220. HOCKING; R. R. & LESLIE R. N. (1967) Selection of the best subset in regression analysis. 7’rc,/?rlor,l~lrit,.s 9. 53 I-540. JANSON C. A. & CLLLANI) W. W. (1974) The kinetic mechanism of glycerokinase. J. hiol. Chr,r~~.249. 2562 7566. JEDZINIAK J. A.. CHYLA(.K L. T.. CH~N(; H-M.. GII.I.IS M. K.. KALUSTIAU A. A. & TLN(; W. H. (19X1) The sorbitol pathway in the human lens: Aldose reductase and polyol dehydrogenase. I~IYY~. Op/~r/ltr/. l’is. %i. 20. 3 144326. JIZFFERYJ.. CUMMLYS L.. CARLQLIIST M. & JORI*;VALL H. (1981) Properties of sorbitol dehydrogenasc and characterization of a reactive cysteine residue reveal uncxpetted similarities to alcohol dchydrogcnase. Eur. J. Bio~/IUJJI.120, 219-234. JORNVALL H.. P~RSSO~ M. & JI:FFI:RY J. (19x1) Alcohol and polyol dehydrogenases arc both divided into two protein types, and structural propertics cross-related the diffcrent enzyme activities with each type. Proc. ~I(/~JI.Ac~trd. SC;.. C’.S.A. 78, 4226 4130. LEISSINC; N. & MCGUII\;N~SS E. T. (197X) Rapid affinity purification and properties of rat liver sorbitol dchydrogenase. Biochim. hioph_v.s. Acfrr 524. 254 26 I. PURICH D. L.. ALLISOI\I R. D. & TODHLNT~R J. A. (1977) On the rapid equilibrium assumption and the problem of distinguishing certain ordered and random kinetic mechanisms. Eioc/lc,n~. hioph~s. Rcs. Comnm. 77, 753 759. STAYTON M. M. & FROMM H. J. (1979) Purifcation. properties. and kinetics of I,-ribulokinase from Aerobatter aerogenes. J. bid. C/~em. 254. 3765-377 I, TSAI C. S. (1982) Multifunctionality of liver alcohol dehydrogenase: Kinetic and mechanistic studies of csterase reaction. Arch Biochrrn. Biop/~vs. 213. 635-642. UE~‘C; S. T-H. & MCGLIIKNLSS ‘E. T. 11977) I,-Mannitol dehydrogenase from Absidfrr q/~mcu. Steady-state kinetic properties and the inhibitory role of mannitol I-phosphate. Biochrr~listry 16, 107- I I I. WILKINSON G. N. (1961) Statistical estimations in enzyme kinetics. Bioc~hrrn. J. 80. 324333.