Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate

An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.     

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.     

Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.     

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.     

Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.     

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii.

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.     

Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis.

The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mM for the fast phase, 0.45 mM for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min-1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme-oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the MODELLER 4 program. The model confirmed that Lys360 is positioned at the NAD+-binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360-->Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360-->Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.     

Inactivation of Escherichia coli L-threonine dehydrogenase by 2,3-butanedione. Evidence for a catalytically essential arginine residue

Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics. The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase. Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e. 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection. Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues. In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+. Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site.     

Chemical modification of specific active site amino acid residues of Enterobacter aerogenes glycerol dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (G1DH 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 G1DH indicated the specific modification of epsilon-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 G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 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 G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast

The baker's yeast saccharopine dehydrogenase (EC 1.5.1.7) was inactivated by 2,3-butanedione following pseudo-first-order reaction kinetics. The pseudo-first-order rate constant for inactivation was linearly related to the butanedione concentration, and a value of 7.5 M-1 min-1 was obtained for the second-order rate constant at pH 8.0 and 25 degrees C. Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected. Although as many as eight arginine residues were lost on prolonged incubation with butanedione, only one residue appears to be essential for activity. The modification resulted in the change in Vmax, but not in Km, values for substrates. The inactivation by butanedione was substantially protected by L-leucine, a competitive analogue of substrate lysine, in the presence of reduced nicotinamide adenine dinucleotide (NADH) and alpha-ketoglutarate. Since leucine binds only to the enzyme-NADH-alpha-ketoglutarate complex, the result suggests that an arginine residue located near the binding site for the amino acid substrate is modified. Titration with leucine showed that the reaction of butanedione also took place with the enzyme-NADH-alpha-ketoglutarate-leucine complex more slowly than with the free enzyme. The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased. From these results it is concluded that an arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.     

Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.     

Physicochemical evidence for the existence of two pyridoxal 5'-phosphate binding sites on glutamate dehydrogenase and characterization of their functional role.

Kinetic studies of pyridoxal 5'-phosphate binding to glutamate dehydrogenase (EC 1.4.1.3) has provided evidence for two specific binding sites, chemically identified as Lys 126 and Lys 333. Use of protecting ligands permitted the selective modification of only one of these lysines, and showed that (1) Lys 333 modification results in depolymerisation of the enzyme into active hexamers; (2) Lys 126-modified enzyme was 92% inactivated. The residual activity was desensitized to GTP. The inactivation process was cooperative, maximum inactivation occurring as soon as half of the Lys 126 were modified.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations.

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.     

Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli

NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics. The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol. The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent. In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.     

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.     

Selective inactivation of various acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA

Inactivation of five distinct acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), the toxic metabolite of hypoglycin from unripe ackee fruit, was investigated using purified enzyme preparations. Short-chain acyl-CoA (SCADH), medium-chain acyl-CoA (MCADH) and isovaleryl-CoA (IVDH) dehydrogenases were severely and irreversibly inactivated by MCPA-CoA, while 2-methyl-branched chain acyl-CoA dehydrogenase (2-meBCADH) was only slowly and mildly inactivated. Long-chain acyl-CoA dehydrogenase (LCADH) was not significantly inactivated, even after prolonged incubation with MCPA-CoA. Inactivation of SCADH, MCADH and IVDH was effectively prevented by the addition of substrate. This mode of inactivation by MCPA-CoA explains the urinary metabolite profile in hypoglycin treated-rats, which includes large amounts of metabolites from fatty acids and leucine, and relatively small amounts of those from valine and isoleucine. Spectrophotometric titration of SCADH and MCADH with MCPA-CoA, together with the protective effects of substrate, indicates that MCPA-CoA is acted upon by, and exerts in turn irreversible inactivation of, SCADH and MCADH, confirming that MCPA-CoA is a suicide inhibitor (Wenz et al. (1981) J. Biol. Chem. 256, 9809-9812). Spectrophotometric titration data of LCADH and MCPA-CoA is typical of non-reacting CoA ester.     

Modification of pig M4 lactate dehydrogenase by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.     

Fluorescence depolarization study of randomly oriented and magneto-oriented spinach chloroplasts in suspension

The sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (GPD) which catalyzes the hydrolysis of acyl phosphates is inactivated by fairly high concentrations of benzylamine. During the inactivation, ¹⁴C-benzylamine is incorporated into the oxidized enzyme. The amount of radioactivity incorporated is nearly stoichiometric with the degree of inactivation of acyl phosphatase activity. Benzylamine does not inactivate the dehydrogenase activity of reduced GPD. Treatment of oxidized GPD with dithiothreitol after it has been partly inactivated with ¹⁴C-benzylamine decreases the amount of radioactivity bound to the enzyme. This evidence is consistent with the reaction of benzylamine with the sulfenic at the active site of oxidized GPD to form a sulfenamide derivative of the enzyme     

Properties of horse liver alcohol dehydrogenase modified by the affinity label 3-chloroacetylpyridine-adenine dinucleotide

The reactive analogue of NAD+, CPAD+, was incorporated in the horse liver alcohol dehydrogenase (EC 1.1.1.1) linearly with its inactivation, to stoichiometry, with no apparent subunit interaction. No hydride transfer could take place in the modified enzyme, nor the interaction of trans-4-N,N-dimethylaminocinnamaldehyde with its reduced form, indicating an impairment of the accessibility to the catalytic zinc atom. The labeling in the enzyme, alkylated by [carbonyl-14C]CPAD+ was not stable, with a half-life of 32 h.