Ferrochelatase from Rhodopseudomonas sphaeroides: substrate specificity and role of sulfhydryl and arginyl residues.

Purified ferrochelatase (protoheme ferrolyase; EC 4.99.1.1) from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the Km for ferrous iron was not altered but that the Km for the porphyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. We also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. We found that 2,4-bis-acetal- and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Our data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme.     

Involvement of arginine residues in glutathione binding to yeast glyoxalase I

Yeast glyoxalase I was inactivated by arginine-specific reagents. Inactivation by 2,3-butanedione, phenylglyoxal and camphorquinone 10-sulfonic acid followed pseudo first-order kinetics with the rate dependent upon modifier concentration. Extrapolation to complete inactivation showed modification of approx. two of the ten total arginyl residues in the native enzyme, with approx. one residue protected by glutathione (GSH) as determined by [ring-14C]phenylglyoxal incorporation. GSH protected the enzyme from inactivation, whereas methylglyoxal, glutathione disulfide (GSSG) and dithiothreitol afforded partial protection. The hemimercaptal of methylglyoxal and GSH and the catalytic product, S-lactoylglutathione provided substantial protection from inactivation. A methyl ester placed on the glycyl carboxyl moiety of GSH abolished all protective capability which suggests that this functionality is responsible for binding to the enzyme. These results provide the first evidence concerning the molecular binding mode of GSH to an enzyme. Arginyl residues are proposed as anionic recognition sites for glutathione on other GSH-utilizing enzymes.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Reactivity and specificity of alpha-dicarbonyl compounds towards essential aminoacyl residues of D-beta-hydroxybutyrate dehydrogenase from the inner mitochondrial membrane

The effects of a alpha-dicarbonyl chromophoric reagent: 4-hydroxy-3-nitrophenylglyoxal on the D-beta-hydroxybutyrate dehydrogenase have been compared to those of phenylglyoxal, a specific arginyl reagent in proteins. Both reagents inactivate irreversibly the enzyme. Kinetic experiments show that only one molecule of these reagents per molecule of enzyme is sufficient to inactivate the enzyme. The second order inactivation rate constant is more than 500 times higher with the chromophoric reagent than with phenylglyoxal. A pseudosubstrate (methylmalonate) in presence of coenzyme (NAD) strongly protects enzyme against inactivation by both reagents. Coenzyme alone has no effect on inactivation by phenylglyoxal while it protects whether inhibitor is the chromophoric reagent or N-ethylmaleimide: a thiol specific reagent. These results indicate: 1. That one arginyl residue is essential for D-beta-hydroxybutyrate dehydrogenase activity (experiments with phenylglyoxal). 2. That the presence of a nitro group on position 3 and a hydroxyl-group on position 4 strongly increase the reactivity of the alpha-dicarbonyl groups, but the specificity of the chemical reaction with arginyl residues seems to be lost for the benefit of cysteyl residues.     

Modification of arginine residues in pyruvate kinase (author's transl)

Pyruvate kinase from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. The loss of activity is caused by the reaction of a single molecule of phenylglyoxal per subunit of enzyme. During inactivation 3 - 6 arginyl residues are modified dependent on the concentration of phenylglyoxal used for modification. The solubility of the protein is reduced by the modification. ATP or phosphoenolpyruvate protect against inactivation. A single arginine is less subject to chemical modification in their presence. Therefore we assume that an arginine is essential at the substrate binding site. The activating ion K does not affectinactivation, where as Mg2 diminishes inactivation. Pyruvate kinase from rabbit muscle is modified by phenylglyoxal in a similar manner.     

Control of phosphoribosylpyrophosphate synthesis in human lymphocytes.

In an attempt to determine if arginyl residues play a role in sulfate transfer reactions, we studied the effects of 2,3-butanedione and phenylglyoxal, both chemical modifying agents for arginyl residues, on phenol-sulfotransferase. Both reagents produced rapid inactivation of the enzyme, with the inactivation following pseudo-first order kinetics. The rate of inactivation was dependent upon the concentration of the chemical modifier. Competition studies showed that inclusion of 3′-phosphoadenosine-5′-phosphosulfate during the preincubation step protected the enzyme from inactivation. The results suggest a possible role for arginyl residues as anionic recognition sites for sulfate transfer reactions.     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.     

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.     

Reaction of phenylglyoxal and (p-hydroxyphenyl) glyoxal with arginines and cysteines in the alpha subunit of tryptophan synthase

The alpha subunit of tryptophan synthase from Escherichia coli is inactivated by phenylglyoxal and by (p-hydroxyphenyl)glyoxal. The use of these chemical modification reagents to determine the role of arginyl residues in the alpha subunit of tryptophan synthase has been complicated by our finding that these reagents react with sulfhydryl groups of the alpha subunit, as well as with arginyl residues. Analyses of the data for incorporation of phenyl[2-14C]glyoxal, for inactivation, and for sulfhydryl modification in the presence and absence of indole-3-glycerol phosphate indicate that two sulfhydryl groups and one arginine are essential for the activity. Our finding that the substrate protects the single essential arginyl residue but not the two sulfhydryl groups is consistent with the observed kinetics of partial protection by substrate or by a substrate analogue, indole-3-propanol phosphate. In contrast to phenylglyoxal, (p-hydroxyphenyl)glyoxal modifies two to three sulhydryl groups that are not protected by indole-3-glycerol phosphate and modifies none of the arginyl residues that are modified by phenylglyoxal.     

Chemical modification of arginine residues of rat liver S-adenosylhomocysteinase

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by phenylglyoxal following pseudo-first order kinetics. The dependence of the apparent first order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is second order in reagent. This fact together with the reversibility of inactivation upon removal of excess reagent and the lack of reaction at residues other than arginine as revealed by amino acid analysis and incorporation of phenylglyoxal into the protein indicate that the inactivation is due to the modification of arginine residue. The substrate adenosine largely but not completely protects the enzyme against inactivation. Although the modification of two arginine residues/subunit is required for complete inactivation, the relationship between loss of enzyme activity and the number of arginine residues modified, and the comparison of the numbers of phenylglyoxal incorporated into the enzyme in the presence and absence of adenosine indicate that one residue which reacts very rapidly with the reagent compared with the other is critical for activity. Although the phenylglyoxal treatment does not result in alteration of the molecular size of the enzyme or dissociation of the bound NAD+, the intrinsic protein fluorescence is largely lost upon modification. The equilibrium binding study shows that the modified enzyme apparently fails to bind adenosine.     

Arginyl residues are involved in the transport of Fe2+ through the plasma membrane of the mammalian reticulocyte

Sealed reticulocyte ghosts were treated with reagents that modify a variety of amino acid residues. Only ninhydrin and phenylglyoxal, both modifiers of arginyl residues, produced inhibition of the initial rate of ⁵⁹Fe²⁺ uptake. The inhibition (i) was dependent on the concentration of ninhydrin or phenylglyoxal, (ii) increased from pH 7 to 9, a feature of the modification of arginine by ninhydrin or phenylglyoxal, and (iii) was blocked when Fe²⁺ was present during the modification step. A23187, an effective membrane Fe²⁺ transporter, diminished the inhibitory effect of ninhydrin and phenylglyoxal, indicative that the transport of iron through the membrane, and not a secondary process, was selectively inhibited. We conclude that the iron transporter from the plasma membrane of erythroid cells has one or more arginyl residues in a segment accessible to ninhydrin or phenylglyoxal, and that this residue is involved in the transmembrane transport of iron.This work was supported by grant 1080-91 from FONDECYT, Chile.     

Implication of arginyl residues in mRNA binding to ribosomes

Modification of Escherichia coli robosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)-dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.     

The binding mechanism of glutathione and the anti-tumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125;NSC-163501) to gamma-glutamyltransferase

The glutathione-protein binding interactions of rat renal gamma-glutamyltransferase (gamma GT) were studied by examining the effect of phenylglyoxal (PGO), a chemical modifying agent for arginyl residues. PGO inactivation of gamma GT followed pseudo-first order kinetics and the rate was dependent upon the concentration of PGO. Glutathione (GSH) protected the enzyme from inactivation by PGO. The anti-tumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) inactivated purified gamma GT. The inactivation capability of AT-125 was abolished by esterification of the carboxyl moiety and was regained upon incubation of AT-125 methyl ester with a carboxyl esterase. AT-125 and glutathione may bind to gamma GT via the electrostatic interaction of their respective carboxyl group(s) and an arginyl residue at the active site.     

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.     

Comparative titration of arginyl residues in purified D-β-hydroxybutyrate apodehydrogenase and in the reconstituted phospholipid-enzyme complex

In order to titrate and understand the role of arginyl residues of D-β-hydroxybutyrate dehydrogenase, arginyl specific reagents: butanedione, 1,2-cyclohexanedione and phenylglyoxal were incubated with three different forms of the enzyme; native enzyme (inner mitochondrial membrane bound), purified apoenzyme (phospholipid -free) and phospholipid-enzyme complex (reconstituted active form).After complete inactivation of the enzyme by [¹⁴C]-phenylglyoxal, the number of modified arginyl residues was different: one with the lipid-free apoenzyme and three with the phospholipid-enzyme complex, suggesting a conformational change of the enzyme triggered by the presence of phospholipids.After exhaustive chemical modification either of the apoenzyme or of the phospholipid-enzyme complex with [¹⁴C]-phenylglyoxal, four arginyl residues were titrated indicating that these residues are located in the hydrophilic part of the enzyme, not interacting with phospholipids.Reconstituted enzyme inactivated by butanedione could no longer bind a pseudosubstrate (succinate) which indicates that an arginyl residue is involved in the enzyme-substrate complex formation.The values of second order rate constants of D-β-hydroxybutyrate dehydrogenase inactivation by butanedione and 1,2-cyclohexanedione were unchanged with the three enzyme forms, suggesting that phospholipids are not involved in the substrate binding mechanism.     

Evidence for an essential arginine residue in the active site of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. Modification with 1,2-cyclohexanedione

Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order. Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log [cyclohexanedione] gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme. The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase. The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues. Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate. In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect. These results indicate that an arginine residue is required for E. coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.     

Presence of an essential arginyl residue in D-β-hydroxybutyrate dehydrogenase from mitochondrial inner membrane

D-β-hydroxybutyrate dehydrogenase, a lipid requiring enzyme, is rapidly and completely inactivated by phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione. Inactivation, which occurs at the millimolar range, depends on the nature of buffer, borate ions are required to get enzyme inactivation by 2,3-butanedione. Most of the inactivation follows a pseudo first order kinetics, the stoichiometry being of one to one. Presence of NAD⁺ or methylmalonate (a substrate-like compound) prior addition of inhibitor does not affect inactivation, while methylmalonate in presence of NAD⁺ strongly protects against inactivation. Chemical modification of the enzyme does not affect K_D of NAD while K_M of β-hydroxybutyrate and Ki of methylmalonate (protecting agent) increase. In view of the high specificity of these inhibitors for arginyl residues of proteins, these results are in favour of the presence of at least one arginyl residue essential for enzyme activity and located in, or near the substrate binding site.     

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

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.