The kinetic properties of spinach leaf glyoxylic acid reductase.

Abstract The kinetic properties of spinach leaf glyoxylic acid reductase have been evaluated. In the presence of pyridine nucleotides, the enzyme catalyzes the reversible reduction of glyoxylate to glycolate and hydroxypyruvate to d(-)-glycerate. The pH optima of the reductive reactions are between 6.0 and 6.5; the pH optima of the oxidative reactions are at 8.9. The enzyme is competitively inhibited by pyruvate, 3-mercaptopyruvate, and 3-fluoropyruvate and noncompetitively inhibited by dihydroxyfumarate. A spectral shift in protein absorbance, from 280 mµ to 270 mµ, is associated with the dihydroxyfumarate inhibition. Ternary rate equations of the enzyme and a degraded form of the enzyme have been calculated and compared. Specific changes in the Michaelis constant for pyridine nucleotides suggest that the degraded form has undergone structural modifications in the pyridine nucleotide binding site. All forms of the enzyme are anion-regulated in the direction of d(-)-glycerate or glycolate formation. Phosphate stimulates hydroxypyruvate reduction at all anion concentrations; chloride, bromide, sulfate, and nitrate stimulate at low concentrations but inhibit at high. The anion effects are pH-dependent and are competitive with hydroxypyruvate or glyoxylate. They are noncompetitive with reduced pyridine nucleotides. Evidence is presented which suggests that a ligand-induced conformational change is important in the functioning of anion effectors.

In the presence of pyridine nucleotides, the enzyme catalyzes the reversible reduction of glyoxylate to glycolate and hydroxypyruvate to D( -)glycerate.
The pH optima of the reductive reactions are between 6.0 and 6.5 ; the pH optima of the oxidative reactions are at 8.9. The enzyme is competitively inhibited by pyruvate, 3-mercaptopyruvate, and 3-fluoropyruvate and noncompetitively inhibited by dihydroxyfumarate. A spectral shift in protein absorbance, from 280 mp to 270 mp, is associated with the dihydroxyfumarate inhibition. Ternary rate equations of the enzyme and a degraded form of the enzyme have been calculated and compared.
Specific changes in the Michaelis constant for pyridine nucleotides suggest that the degraded form has undergone structural modifications in the pyridine nucleotide binding site. All forms of the enzyme are anion-regulated in the direction of D( -)-glycerate or glycolate formation. Phosphate stimulates hydroxypyruvate reduction at all anion concentrations; chloride, bromide, sulfate, and nitrate stimulate at low concentrations but inhibit at high. The anion effects are pH-dependent and are competitive with hydroxypyruvate or glyoxylate.
They are noncompetitive with reduced pyridine nucleotides.
Evidence is presented which suggests that a ligand-induced conformational change is important in the functioning of anion effecters.
A homogeneous form of spinach leaf glyoxylic acid reductase has been isolated from commercial crystalline preparations (1). The enzyme catalyzes the reduction of hydroxypyruvate as well as glyoxylate and has a molecular weight of 97,500 f 5,000. It is composed of two equal-sized subunits which are dissociated by exposure to 6.0 M guanidine hydrochloride-O.1 M mercaptoethanol, 8.0 ~1 urea-O.1 M mercaptoethanol, or iodoacetate. Analytical disc gel analyses, amino-terminal end group analyses, and peptide mapping indicate that the polypeptide chains of each subunit are identical or very similar in their primary structure.
The subunits can reassociate to yield the native enzyme dimer and a nearly inactive enzyme tetramer.
The present report characterizes the kinetic properties of this enzyme.
It shows that the products of hydroxypyruvate and glyoxylate reduction are D( -)-glycerate and glycolate, respectively.
Anion modification of the reductive reaction is shown and a mechanism for these effects is suggested.
Noncompetitive inhibition by solutions of dihydroxyfumarate is indicated.1 The data are compared with results obtained from similar evaluations of several bacterial "hydroxypyruvate reductases" (2-4) and a tobacco leaf glyoxylate reductase purified by Zelitch (5). The kinetic properties of the enzyme are also compared to the kinetic properties of three additional forms of the protein which have been isolated and characterized (1). Two of these forms are considered isozymic in nature since they have the same molecular weight and amino acid composition as the enzyme above. The third is concluded to be a degraded form of the enzyme since it has a lower molecular weight and a different amino acid content (1). These forms have been shown to be readily distinguished by their specific activities, isoelectric points, and stabilities.

MATERIALS AND METHODS
All chemicals were obtained from commercial sources or were prepared as described (1). Procedures are noted in context. One unit of enzyme activity is defined as that quantity of 1 Dihydroxyfumarate solutions have been shown to inhibit several other anion-modulated reductases which convert hydroxypyruvate to n (-) -glycerate (24).
Although dihydroxyf umarate solutions contain small amounts of the keto isomer, oxaloglycolate, the enol form is the presumed inhibitor since one of these enzymes (4) can use the keto isomer as its substrate. enzyme catalyzing the formation of 1.0 pmole of reduced or oxidized pyridine nucleotide per min in the standard assay mixtures. Specific activity is defined as units per mg of protein; protein was determined calorimetrically (6) with crystalline bovine serum albumin as the standard.
Kinetic studies used a Beckman model DU spectrophotometer equipped with a Gilford cuvette changer or a Cary model 14 recording spectrophotometer. Cells had a l-cm light path. Control cuvettes contained no enzyme or boiled enzyme, and all studies utilized stock enzyme solutions which were diluted 1 hour before use into potassium phosphate, pH 7.0, containing 0.1% bovine serum albumin (w/v). Thus diluted, the enzymes were stable for 24 hours (1). Albumin did not affect the kinetic data.
Enzyme Preparation-The homogenous enzymes utilized in these studies were obtained from crystalline preparations of spinach leaf glyoxylic acid reductase (Boehringer Mannheim). They were isolated by gel filtration chromatography, preparative gel electrophoresis, or isoelectric focusing as described (1) TPNH replaced DPNH as the reduced pyridine nucleotide, but not readily. At pH 6.4 and in the presence of 0.15 mu TPNH, the K, value for hydroxypyruvate was 1.4 x l&* M but the K, value for glyoxylate could not be calculated. In the presence of 3 mM hydroxypyruvate or 50 mM glyoxylate, the K, values for TPNH were 4 X 10B4 M and 1 x 1e4 M, respectively. Maximal velocity values were at least IO-fold lower than those obtained in the presence of DPNH.
Kinetic analysis (7) of the reduction of glyoxylate or hydroxypyruvate by DPNH allowed the determination of the constants, K,, Kb, and K,, for the general rate equation (Equation 1). In this equation, V,, designates the maximum velocity at a given enzyme concentration and v represents the observed velocity. K, and & may be considered "limiting Michaelis constants" for the Substrate A and B (7). K, is an interaction constant describing the kinetic effects of having both substrates on the enzyme at one time (7). Data for all studies were obtained for initial rates of reaction, i.e. before 10% of the substrates had been utilized. [DPNHJ In Equations 2 and 3, respectively, values of the constants for the reduction of glyoxylate and hydroxypyruvate are presented. In these and subsequent equations, the values are the average of at least three separate experiments on different lots of enzyme and different batches of substrates. Experimental values were in satisfactory agreement with the curves calculated from these equations (Fig. 1). The results implicate the existence of a ternary complex involving enzyme, carbonyl substrate, and pyridine nucleotide but do not exclude a Theorell-Chance mechanism (7,8).
Enzyme concentrations used to obtain data for Equation 2 were lo-fold higher than concentrations of enzyme needed to evaluate Equation 3. At the same enzyme concentration, the maximal velocity (V,,,) of hydroxypyruvate reduction was 3.7fold greater than that for glyoxylate catalysis.
Product--D( -)-Glyceric acid was identified as the product when the enzyme catalyzed the reduction of hydroxypyruvate; glycolic acid was produced from glyoxylate.
For the isolation of products, 3-ml cuvettes having a l-cm light path were maintained at 25" and were monitored continuously by measurement of absorbance at 340 rnp. DPNH was the reduced pyridine nucleotide in all experiments; control cuvettes were identical with experimental ones, except for the substitution of boiled enzyme for active enzyme.
Four cuvettes were used to isolate the product of hydroxypyruvate reduction. In a total volume of 2 ml, each cuvette contained potassium phosphate at pH 6.4, 200 pmoles; lithium hydroxypyruvate, 150 pmoles; and enzyme, 0.05 mg. Over the course of 1 hour, 75 pmoles of reduced pyridine nucleotide were added to each cuvette in 5-to lO+mole increments. Reactions were terminated by acidification to pH 2.0 with sulfuric acid. To monitor the isolation, tracer amounts of nn-glyceric acid-1-14C (14 mCi per mmole) were added to one cuvette and the reaction mixtures were pooled, heated, charcoal-treated, absorbed to columns of Dowex l-acetate, and eluted as previously described (2)(3)(4). Radioactivity was detected in the eluate collected between 190 and 248 ml. Concentrated to 2 ml in a rotary evaporator maintained at 40", the solution was assayed for glyceric acid by means of the chromotropic acid reaction with (9) or without (10) prior periodate treatment. A yield of 232 pmoles of glyceric acid was obtained, no glyceric acid being detected in control samples. Since the average recovery of radioactivity was 82% and since the radioactive glyceric acid had been found to be chromatographically pure, it was calculated that 283 pmoles of glycerate were produced from 300 pmoles of DPNH which had been observed, spectrophotometrically, to be oxidized.
The ? for several samples was between +111.2 and 120.8 when calculated for the sodium salt (24). At pH 8.5, in the presence of 2 ITIM DPN, 0.1 M hydrazine, and the isolated product, lactic dehydrogenase did not cause an increase in 340 rnp absorbance; hydroxypyruvate reductase (2) did.
For the determination of the product of glyoxylate reduction experiments were identical with those above with the following modifications: the incubation mixtures contained 40 pmoles of potassium phosphate, pH 6.4, and 500 pmoles of glyoxylate; the heating step was eliminated; and glycolic acidJ4C was used as the marker. Glycolic acid was eluted from the ion exchange columns between 150 and 180 ml (24) and was assayed by the method of Calkins (11). After 2 to 3 hours of incubation, 79 pmoles of DPNH were oxidized, 58 pmoles of glycolic acid were isolated, and 80% recoveries were determined. Enzyme concentration for the glyoxylate kinetic studies was 0.228 pg per ml; enzyme concentrations for the hydroxypyruvate kinetics were lo-fold lower.
The reaction was reversible with D( -)-glyceric acid in the presence of DPN. It was linear with respect to time and proportional to protein concentration. At 25", an equilibrium constant of 1.61 x 1012 was calculated from the data in Table I and from Equation 4. This represents a AF of -16.7 kcal per mole at 25" and of -7.1 kcal per mole at pH 7.0 at the same temperature.
In the presence of DPN, the reaction was also reversible with glycolic acid, and at 25" an equilibrium constant of 3.83 X 1014 was calculated from the data in Table II and from Equation 5.
This represents a AF of -19.9 kcal per mole at 25" and of - 10.4 kcal per mole at pH 7.0 at the same temperature.
[  of Reverse Reaction-In Tris-chloride at pH 8.5, ternary kinetics was again observed and the following rate equations could be calculated for D( -)-glycerate or glycolic acid oxidation (Equations 6 and 7). In both cases, experimental data were in good agreement with the curves generated from these rate equations (Fig. 2) The relative maximal velocity of the forward and reverse reactions was 30-fold greater in the direction of glycerate or glycolate formation under the stated conditions.
In the presence of 2 InM DPN and at pH 8.5 in Tris-chloride tartrate, or mesotartrate. pH Optima-With standard assay conditions, the optimal pH for the reduction of glyoxylate was between 5.5 and 6.5 (Fig.  3A). For the reduction of hydroxypyruvate, the optimal activity was from pH 6.1 to 6.6 (Fig. 3B), and for the oxidation of glycolate or glycerate it was at pH 8.9 (Fig. 3C). Sensitivity of the pH curves to changes in the buffer concentration was noted and can be seen in Fig. 3B. Enzyme activity decreased much more sharply between pH 6.5 and 8.0 when the phosphate concentration was increased from 0.06 M to 0.1 M. E$ect of SpeciTc Anions and Anion Concentration on Enzyme Activzty-As with hydroxypyruvate reductase (2), tartronic semialdehyde reductase (3), and oxaloglycolate reductive decarboxylase (4), the activity of the enzyme was markedly dependent upon the presence of various anions. With DPNH and hydroxypyruvate as substrates, the enzyme showed an optimum anion concentration beyond which inactivation occurred (Fig. 41). When glyoxylate was the carbonyl substrate, the activation phase was barely discernible with most anions (Fig. 411) and the optimal enzymatic activity was obtained at lower anion concentrations.
The most effective activator was phosphate and the most effective inhibitor was Concentrations corresponding to zero salt represent basal buffer concentrations of either 25 mu potassium phosphate (1) or 10 mM potassium phosphate (II).
Standard assay conditions were otherwise maintained. With the exception of ammonium bromide (A), for which only the noted cation was used, essentially identical curves were obtained with the ammonium, potassium, and sodium salts of the following anions; phosphate (O), sulfate (0), chloride (A), and nitrate (m). The pH was between 6.35 and 6.45 in each experiment.
nitrate. Tobacco leaf glyoxylate reductase, which was not inhibited by anions (5), had a reversed anion modulation; i.e. the most effective activator was nitrate and the least effective phosphate.
The salt effects were pH-dependent at both high and low anion concentrations.
As can be seen in Fig. 5, where the data are plotted as double reciprocal kinetic studies, the effect at high anion concentrations was predominantly on the V,, whereas at low concentrations the effect was predominantly at the K,; i.e. there was negative cooperativity (12). When nitrate, chloride, or sulfate anions were present, the data were similarly pHdependent, although no activation was observed when nitrate concentrations were in the high salt range, i.e. greater than 50 mu. Activation at low anion concentrations again occurred without changes in enzyme maximal velocity. of potassium phosphate, pH 6.4. Sephadex G-25 columns, 2 X 15 cm, were used; protein load was 2.5 mg. Protein was tritiated and equilibrated in 7.5 mM potassium phosphate, pH 6.4, before gel filtration.
The temperature was 25" and the flow rate was 1 ml per min. Fractions of 2 ml were collected.
Columns run at 50 mM, 100 mM, and 150 mu potassium phosphate were the same as the column run at 40 mM potassium phosphate.
(0.01 to 0.2 M potassium phosphate at pH 6.4 or pH 6.0) ; however, by using tritium labeling and gel filtration chromatography (13), effects of ionic strength on secondary structure (14, 15) could be shown. Glyoxylic acid reductase was equilibrated with 0.01 M Tris-chloride, pH 7.3 (low salt concentration), or with 0.2 M Tris-chloride, pH 7.3 (high salt concentration), or both, by dialysis for 24 hours in a 6000-fold excess of buffer. Ten microliters of tritiated water (100 mCi per g) were added to 5-mg aliquots of the enzyme and the solutions were incubated for several days at O-2" in order to establish the hydrogentritium exchange equilibrium. Aliquots of the labeled proteins were applied to columns (2 x 15 cm) of Sepha.dex G-25 and eluted at a flow rate of 1 ml per min; one column was equilibrated with 0.01 M Tris-chloride at pH 7.3 and the other with 0.2 M Tris-chloride at pH 7.3. Effluent samples were evaluated for enzymatic activity, 280 rnp absorbance, and radioactivity; the counting techniques and corrections were the same as those described (13 9. Absorption spectrum of glyoxylic acid reductase in the presence (---) and absence (-) of dihydroxyfumarate, 1 X lo-' M.
The buffer is 0.1 M potassium phosphate at pH 7.0. Control cuvettes contained buffer or buffer plus dihydroxyfumarate, 1 X 10e4 M, as appropriate.
The effect on native enzyme, RF 0.22, was the same as on the degraded isozyme, RF 0.27.
As can be seen in Fig. 7, A and B, exposure of the tritiated proteins to low salt conditions resulted in a high exchange of label with the medium regardless of the initial conditions of incubation.
Exposure to conditions of high ionic strength resulted in a very much lower exchange of label (Fig. 7, C and D). Similar data were obtained with these buffers in the presence of 20% glycerol (v/v) and in the presence of 0.01 M and 0.2 M potassium phosphate at pH 6.4. The transition between the high and low rate of exchange seemed to lie between 20 and 35 mM potassium phosphate (Fig. 8).
Enzyme Inhibition-With hydroxypyruvate as substrate, the reductive reaction was inhibited in a competitive manner by glyoxylate.
The Ki values for these compounds are presented in Table III.
Associated with dihydroxyfumarate inhibition there was a change in the absorption spectrum of the enzyme, the absorption maximum-shifting from 280 to 270 rnp (Fig. 9). After passage of the dihydroxyfumarate-enzyme solutions through Sephadex G-25 columns, the spectrum reverted to that of the native enzyme, as did the specific activities and kinetics.
The spectral change occurring in the presence of dihydroxyfumarate was the same as had been observed with hydroxypyruvate reductase (2) and tartronic semialdehyde reductase (3), enzymes which yield D( -)-glycerate from the catalytic reduction of hydroxypyruvate.
In contrast, enzymes such as lactic and alcohol dehydrogenase which catalyze the reduction of hydroxypyruvate to L( +)-glycerate, were not inhibited by dihydroxyfumarate and do not show a spectral shift in its presence. Similarly, 3-phosphoglycerate dehydrogenase and malic dehydrogenase are unaffected by dihydroxyfumarate.
No significant inhibition of the enzyme was found at 1 m&r concentrations of sodium EDTA or potassium arsenite.
The reverse reaction, the oxidation of D( -)-glycerate or glycolate, was competitively inhibited by lactate, phosphoglycerate, and propionic acid; however, inhibition was less than 20% at 10 mnr concentrations of each compound.

Kinetic Properties of Other Forms of Glyoxylic Acid Reductase
Degraded Form-This form of the enzyme, RF 0.27 on analytical gels, had a molecular weight of 83,000 f 5,000 and was composed of two subunits, molecular weight 40,000 f 3,000 (1). Peptide mapping studies suggested that this species was a degraded product of the major enzyme, RF 0.22, described above.
The protein catalyzed the reduction of hydroxypyruvate and glyoxylate in the presence of DPNH.
In the presence of 0.15 m&r reduced pyridine nucleotide, compounds which were not substrates with the RF 0.22 native protein did not serve as substrates with this isozyme; the concentrations of the compounds tested were the same as those previously described.
At pH 6.4 and in the presence of 3 MM hydroxypyruvate, the K, value for DPNH was 1.8 X 10T6 M; in the presence of 50 mM hydroxypyruvate, the K, value for DPNH was 2.5 X 10m6 M. With 0.15 M DPNH, the K, values for hydroxypyruvate and glyoxylate were 5 X lop5 and 5 X 10V2 M, respectively.
Kinetic analyses (7) of the reduction of hydroxypyruvate or glyoxylate in the presence of DPNH allowed the calculation of the rate equations below (Equations 8 and 9). The experimental values were in satisfactory agreement with the curves calculated from these equations and again suggested that a ternary complex (7,8)  Comparisons of the kinetics of this isozyme with the kinetics of the Rp 0.22 enzyme described above revealed several consistent relationships.
First, there was no significant difference in maximal velocity (V,,,) between the two proteins when the same substrates were utilized and when similar enzyme concentrations were present; i.e. the Tr,,, terms of Equations 2 and 8, of Equations 3 and 9, etc., were identical when compared.
Second, the V in the presence of hydroxypyruvate was 3-to 4-fold higher thl:the V max obtained when glyoxylate was substrate, independent of the enzyme forms. Last, there was no significant difference in the K, terms, the limiting Michaelis constants, between the two proteins but there was a consistent 2-fold difference in the Kb terms; i.e. the Ka value for DPNH was 3.4 X lo+ as opposed to 1.3 X 10-C in Equations 2 and 8, respectively, and was 5.2 x 10V6 as opposed to 1.9 x 10e6 in Equations 3 and 9, respectively, etc.
The specificity of the reverse reaction, the oxidation of D( -)glycerate or glycolate, was the same for this enzyme form and for the Rp 0.22 enzyme.
Inhibition properties were essentially identical (Table III) and dihydroxyfumarate solutions induced a shift in the absorption spectra analogous to that seen in the RF 0.22 enzyme (Fig. 9). The pH dependence, the salt effects, and the tritium exchange data were also not significantly different from those described for the native enzyme.
Isozymes RF 0.19 and O.i7-Although similar in molecular weight, amino acid composition, and subunit structure, these isozymes could be distinguished from each other by their specific activities and kinetic properties.
The RF 0.19 isozyme was identical with the RF 0.22 enzyme in all respects, i.e. in its substrate specificity, pH optima, salt effects, ternary kinetics, and rate equations.
The RF 0.17 isozyme was also identical with the Rp 0.22 enzyme in its substrate specificity, pH optima, and salt effects; however, distinct differences were evident in its rate equations. Maximal velocity values were Z-to 3-fold lower, and the K, values for hydroxypyruvate, glyoxylate, D( -)-glycerate, and glycolate were 2-fold higher. The Kb values were similar in all rate equations. Double reciprocal plots readily show these differences (Fig. 10). DISCUSSION Spinach leaf glyoxylic acid reductase is similar to several bacterial enzymes capable of the catalytic reduction of hydroxypyruvate, i.e. hydroxypyruvate reductase (2), tartronic semialdehyde reductase (3), and oxaloglycolate-reductive decarboxylase (4). Like these enzymes, it catalyzes the formation of D( -)-glycerate from hydroxypyruvate, is anion-regulated, and is inhibited by dihydroxyfumarate.
The dihydroxyfumarate inhibition is noncompetitive and is associated with the same spectral shift previously observed with bacterial preparations.
Tobacco leaf glyoxylic acid reductase, an enzyme previously described (5), also catalyzes the reversible reduction of glyoxylate and hydroxypyruvate.
Although dihydroxyfumarate inhibition has not been evaluated, the tobacco preparation yields D( -)-glycerate from hydroxypyruvate and is anion-modulated. It is different from the spinach protein in that arsenite inhibits its enzymatic activity and in that TPNH cannot replace DPNH as the reduced pyridine nucleotide. In addition, the anion modulation involves activation only, nitrate being the most effective activator and phosphate the least; i.e. the order of anion activation is exactly reversed.
The kinetics of spinach leaf glyoxylic acid reductase is ternary; i.e. catalysis occurs only when both substrates are present on the enzyme at the same time (7,8). Although all forms of the spinach enzyme exhibit these kinetic properties, differences which exist in the rate equations suggest specific structural modifications in two of the additional enzyme forms. When compared to the RF 0.22 protein, the low molecular weight enzyme, RF 0.27, has a 2-fold difference in the limiting Michaelis constant for pyridine nucleotides (Ka) in the absence of a detectable difference in the analogous constant for carbonyl substrates (KG) and in the absence of a change in enzyme maximal velocity (V,,,).
Whether a random or an ordered mechanism (7, 8) is operative, the altered rate constants necessary to produce this difference should reflect a unique DPNH-enzyme interaction, i.e. a structural change in the binding site for pyridine nucleotides. In the same vein, the Rp 0.17 isozyme has an altered Michaelis constant for the carbonyl substrate but no change in the constant for DPNH.
Although this isozyme has a maximal velocity different from that of RF 0.22 enzyme, structural changes should have affected the carbonyl site more than the DPNH site. The data suggest that studies characterizing the structural differences between these forms will offer significant information concerning the active center of the spinach enzyme. Sulfhydryl studies of these forms have already yielded interesting differences in this regard (16).
Anion effects on the spinach enzyme are extremely complex. At low salt concentrations, hydroxypyruvate reduction is activated in a similar quantitative fashion by all anions (Fig. 4). Double reciprocal plots emphasize this since the maximal enzyme velocity is independent of the anion present. As the salt concentration increases, each anion affects the activity differently, i.e. activating further or inhibiting (Figs. 4 and 5). The shift in anion modulation coincides with a change in enzyme maximal velocity and a change in enzyme conformation (Figs. 7 and 8). Similar enzyme changes in the presence of an effector have been termed negative cooperativity (12,17,18), and a ligand-induced conformational change is presumed responsible once the identity of the peptide chains has been shown and when isozyme contamination and pleomorphic forms have been ruled out (12,17,18). Coupling the data and this presumption (12), a model explaining the anion regulation is proposed. The enzyme is presumed to exist in two distinct conformational states which have different maximal velocities. A specific anion binding site is located near the carbonyl substrate site and a positively charged amino acid is present at that point or nearby on the enzyme molecule. The association of the anion and substrate sites is based on the competitive kinetics with carbonyl substrates; the existence of the positively charged residue is implied from electrostatic considerations and from the pH sensitivity of anion modulation. The suggestion that there is only one anion binding site is arbitrary, and the mechanism of the ligand induced conformational change is unknown.
Since the conformational change is independent of pH and since it apparently occurs with all anions and cations tested, changes in salt concentrations are presently presumed to be causal.
In the "low salt configuration," every anion binding to the enzyme will increase catalytic activity by increasing the affinity of the enzyme for its carbonyl substrates.
In the "high salt configuration," every anion will compete with the carbonyl substrate for enzyme attachment since the anion and substrate sites are conformationally different. In both configurations, a decrease in hydrogen ion concentration eliminates the charge on the amino acid residue responsible for anion binding and eliminates the activation and inhibition. In both configurations, the anion with the highest affinity for the enzyme is the worst activator or best inhibitor.
The model is applicable to a consideration of the structurefunction relationships of anion modulation in the bacterial and tobacco enzymes previously mentioned (2)(3)(4)(5). Hydroxypyruvate reductase (2) and tobacco glyoxylic acid reductase (5) are anion-activated only; tartronic semialdehyde reductase (3) is either activated or inhibited by a particular anion, but not both. With no negative cooperativity, these enzymes should have no ligand-induced conformational shift, and tritium exchange experiments should not be affected by increasing salt concentrations. Hydroxypyruvate reductase (2) should exhibit a low salt exchange pattern, whereas tartronic semialdehyde reductase (3) should exhibit a high salt pattern. Since anion modulation of oxaloglycolate reductive decarboxylase is similar to anion regulation of the spinach enzyme, tritium exchange experiments should show a shift between 20 mM and 75 mM salt concentrations. In all cases, the anions should exhibit competitive kinetics when evaluated against carbonyl substrates.