Chemical modification of lysine residues in bacterial formate dehydrogenase

Specific modification of 4.4 lysine residues per molecule of formate dehydrogenase, from the methylotrophic bacterium Achromobacter parvulus I by pyridoxal, results in complete inactivation of the enzyme. The concentration effect of the modifying agent and substrates on the inactivation of formate dehydrogenase has been studied. Coenzymes do not protect the enzyme from inactivation. Complete maintenance of enzyme activity was achieved in the presence of saturating concentrations of the formate and upon formation of the ternary complex, enzyme-NAD-azide. Formate specifically protects two lysine residues per dimer molecule of the enzyme from modification. The presence of one essential lysine residue in the substrate-binding region of the enzyme active site is assumed.     

An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics. The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase. No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme. Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site. Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme. This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site. The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.     

Molecular basis for the transfer of nicotinamide adenine dinucleotide among dehydrogenases

NADH is transferred directly from one dehydrogenase enzyme site to another without intervention of the aqueous solvent whenever the two dehydrogenases are of opposite chiral specificity as regards the C4 H of NADH which is transferred in the catalyzed reduction reaction. When both enzymes catalyze the transfer of hydrogen from the same face of the nicotinamide ring, direct enzyme-enzyme transfer of NADH is not possible [Srivastava, D. K., & Bernhard, S. A. (1984) Biochemistry 23, 4538-4545; Srivastava, D. K., & Bernhard, S. A. (1985) Biochemistry (preceding paper in this issue)]. Utilizing an advanced computer graphics facility, and the known three-dimensional coordinates for three dehydrogenases, we have investigated the feasibility of various aspects of the direct transfer of dinucleotide from the site of one enzyme to the site of the other. The facile passage of the coenzyme through the first enzyme site requires an open protein conformation, characteristic of the apoenzyme rather than the holoenzyme structure. Since two dehydrogenases of the same chirality bind coenzyme in the same conformation, the direct transfer of coenzyme from one site to the other is impossible due to the restriction in molecular rotation of the coenzyme in the path of transfer from one binding site to the other; therefore, coenzyme can only be transferred from one dehydrogenase site to another site via the intermediate dissociation of coenzyme into the aqueous milieu. In contrast, when an A dehydrogenase and a B dehydrogenase are juxtaposed, it is stereochemically feasible to transfer the nicotinamide ring from its specific binding site in one enzyme to the site in the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a histidine and a cysteine residue in the substrate-binding site of yeast alcohol dehydrogenase.

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited by stoicheiometric concentrations of diethyl pyrocarbonate. The inhibition is due to the acylation of a single histidine residue/monomer (mol.wt. 36000). 2. Alcohol dehydrogenase is also inhibited by stoicheiometric amounts of 5,5'-dithiobis-(2-nitrobenzoate), owing to the modification of a single cysteine residue/monomer. 3. Native alcohol dehydrogenase binds two molecules of reduced coenzyme/molecule of enzyme (mol.wt. 144000). 4. Modification of a single histidine residue/monomer by treatment with diethyl pyrocarbonate prevents the binding of acetamide in the ternary complex, enzyme-NADH-acetamede, but does not prevent the binding of NADH to the enzyme. 5. Modification of a single cysteine residue/monomer does not prevent the binding of acetamide to the ternary complex. After the modification of two thiol groups/monomer by treatment with 5,5'-dithiobis-(2-nitrobenzoate), the capacity of enzyme to bind coenzyme in the ternary complex was virtually abolished. 6. From the results presented in this paper we conclude that at least one histidine and one cysteine residue are closely associated in the substrate-binding site of alcohol dehydrogenase.     

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.     

Mechanistic studies on the rat kidney flavoenzyme L-alpha-hydroxy acid oxidase.

The falvoenzyme L-alpha-hydroxy acid oxidase from rat kidney [T.H Cromartie and C.T. Walsh (1975), Biochemistry 14, 2588] fails to catalyze the elimination of HCl form D,L-beta-chlorolactate, although this compound is a substrate for oxidation by the enzyme. Deuterium isotope effects demonstrate that proton removal from the alpha carbon of alpha-hydroxy acids is fully rate limiting, a finding in agreement with observations on L-lactate dehydrogenase from yeast [F. Lederer (1974), Eur. J. Biochem. 46, 393] which also does not promote elimination from D,L-beta-chlorolactate. Both D-alpha-hydroxy acid oxidase were found to be rapidly and irreversibly inactivated by the acetylenic substrate 1-hydroxy-3-butynoate. The partially purified dehydrogenase was observed to be inactivated within 10 min by 6.8 times 10(-8) M hydroxybutynoate. For the more extensively studied oxidase, inactivation was found to occur after 25 catalytic events, inactivation occurring by covalent addition of the inactivator to the coenzyme. A stoichimometry of one molecule of hydroxybutynoate per flavine was found, and the time course of inactivation was unaffected by the presence of thiols. The oxidase could also be inactivated by prolonged incubation of the enzyme with 2-hydroxy-3-butenoate, and inactivation which could be completely prevented by the presence of thiolds. Since the inactivation with hydroxybutenoate also left the flavine coenzyme unaltered, the inactivation was attributed to Michael addition of nucleophiles on the enzyme of the ketobutenoate product. Several 4-alkyl-substitued 2-hydroxy-3-butynoates were also observed to inactivate the oxidase by both coenzyme modification and random addition to the apoenzyme. It is proposed that the inactivation may occur by nucleophilic addition of a C4 allenic carbanion to the oxidized flavine coenzyme.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.     

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.     

Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.     

Chemical modification of the lysine residues of bacterial formate dehydrogenase

Inactivation of formate dehydrogenase by formaldehyde, pyridoxal and pyridoxal phosphate was studied. The effects of concentrations of the modifying agents, substrates, products and inhibitors on the extent of the enzyme inactivation were examined. A complete formate dehydrogenase inactivation by pyridoxal, pyridoxal, phosphate and formaldehyde is achieved by the blocking of 2, 5 and 13 lysine residues per enzyme subunit, respectively. The coenzymes do not protect formate dehydrogenase against inactivation. In the case of modification by pyridoxal and pyridoxal phosphate a complete maintenance of the enzyme activity and specific protection of one lysine residue per enzyme subunit is observed during formation of a binary formate-enzyme complex, or a ternary enzyme--NAD--azide complex. One lysine residue is supposed to be located at the formate-binding site of the formate dehydrogenase active center.     

Evidence for the proximity of two sulfhydryl groups at the active site of 6-phosphogluconate dehydrogenase

The reaction between 6-phosphogluconate dehydrogenase from Candida utilis and 5,5′-dithiobis(2-nitrobenzoate) results in the inactivation of the enzyme. At pH 6.0 the inactivation can be correlated with the modification of only one SH group per enzyme subunit. The modified SH group can react with another SH group forming an intramolecular disulfide bridge. Since the modified enzymes, either with an SH group modified or with a cystine disulfide bridge, are still able to bind the substrate and the coenzyme, gross conformational changes seem unlikely to have occurred. The results obtained suggest that the SH groups of two cysteine residues are located close to each other in the three-dimensional structure of the active site of the enzyme.     

Selective chemical modification of arginine residues in mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart exhibits a time dependent loss in enzymatic activity in the presence of the reagent butanedione. The inhibition occurs concomitant with the modification of 2.4 residues of arginine per molecular weight of 70,000. The presence of the reduced coenzyme, NADH, protects the enzyme from inhibition by butanedione and from modification of arginine residues, suggesting that the residues modified are located near the coenzyme binding site and hence at or near the enzymatic active center of this enzyme.     

Depolymerisation of F-actin to G-actin and its repolymerisation in the presence of analogs of adenosine triphosphate.

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.     

Chemical Modification of Specific Active Site Amino Acid Residues of Enterobacter aerogenes Glycerol Dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (GlDH EC 1.1.1.6), a tetrameric NAD + specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5′-phosphate (PLP) and o -phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced GlDH indicated the specific modification of ? -amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD + and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of GlDH by the bifunctional reagent OPA, which reacts specifically with proximal ? -NH 2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD + was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of GlDH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S-cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.     

Conditions for continuous registration of enzyme activity

Conditions of continuous registration of enzyme activity are considered on the example of alcohol dehydrogenase and sorbitol dehydrogenase from cytoplasm of the bovine liver cells. A device permitting to register the initial steps of enzyme interaction with the effector (substrate, coenzyme or inhibitor) is described. The importance of the reaction product coupling for analysis of enzyme activity is demonstrated.