The presence of essential carboxyl group for binding of cytochrome c in rat hepatic NADPH-cytochrome P-450 reductase by the reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

NADPH-cytochrome P-450 reductase (EC 1.6.2.4) purified from rat hepatic microsomal fraction was inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a specific agent for modification of carboxyl groups in a protein. The inactivation exhibited pseudo-first order kinetics with a reaction order approximately one and a second-order-rate constant of 0.60 M-1 min-1 in a high ionic strength buffer and 0.08 M-1 min-1 in a low ionic strength buffer. By treatment of NADPH-cytochrome P-450 reductase with EDC, the pI value changed to 6.5 from 5.0 for the native enzyme, and the reductase activity for cytochrome c, proteinic substrate, was strongly inactivated. When an inorganic substrate, K3Fe(CN)6, was used for assay of the enzyme activity, however, no significant inactivation by EDC was observed. The rate of inactivation by EDC was markedly but not completely decreased by NADPH. Also, the inactivation was completely prevented by cytochrome c, but not by K3Fe(CN)6 or NADH. The sulfhydryl-blocked enzyme prepared by treatment with 5,5'-dithio-bis(2-nitrobenzoic acid), which had no activity, completely recovered its activity in the presence of dithiothreitol. When the sulfhydryl-blocked enzyme was modified by EDC, the enzyme in which the carboxyl group alone was modified was isolated, and its activity was 35% of the control after treatment with dithiothreitol. In addition, another carboxyl reagent, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K), decreased cytochrome c reductase activity of NADPH-cytochrome P-450 reductase. These results suggest that the carboxyl group of NADPH-cytochrome P-450 reductase from rat liver is located at or near active-site and plays a role in binding of cytochrome c.     

Chemical and kinetic evidence for an essential histidine in horseradish peroxidase for iodide oxidation.

Horseradish peroxidase (HRP), when incubated with diethylpyrocarbonate (DEPC), shows a time-dependent loss of iodide oxidation activity. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.43 min-1 M-1 at 30 degrees C and is reversed by neutralized hydroxylamine. The difference absorption spectrum of the modified versus native enzyme shows a peak at 244 nm, characteristic of N-carbethoxyhistidine, which is diminished by treatment with hydroxylamine. Correlation between the stoichiometry of histidine modification and the extent of inactivation indicates that out of 2 histidine residues modified, one is responsible for inactivation. A plot of the log of the reciprocal half-time of inactivation against log DEPC concentration further suggests that only 1 histidine is involved in catalysis. The rate of inactivation shows a pH dependence with an inflection point at 6.2, indicating histidine derivatization by DEPC. Inactivation due to modification of tyrosine, lysine, or cysteine has been excluded. CD studies reveal no significant change in the protein or heme conformation following DEPC modification. We suggest that a unique histidine residue is required for maximal catalytic activity of HRP for iodide oxidation.     

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.     

Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor

Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.     

The role of cysteinyl residues in the phosphorylase kinase activity as revealed by iodacetamide modification

In native nonactivated phosphorylase kinase [14C] iodacetamide interacts with 50 cysteinyl residues per enzyme molecule (alpha beta gamma delta)4. According to their reactivity towards iodacetamide these residues can be classified into 3 groups. The most reactive cysteinyl residues are involved in the enzyme activation caused by modification of SH-groups. The enzyme inhibition is biphasic. The fast and slow inactivation reactions follow the pseudo-first order kinetics. The rate of inactivation is increased by Ca2+. Mg-ATP effectively protects the enzyme against the inactivation and chemical modification of three SH-groups per protomer (apha beta gamma delta). The kinetics of inactivation and of the [14C] iodacetamide label incorporation demonstrate that two cysteinyl residues per enzyme protomer (alpha beta gamma delta) are essential for the enzyme activity. These residues are located near the ATP-binding site of the beta and gamma subunits of phosphorylase kinase.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide

S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.     

Site-site heterogeneity in isocitrate lyase of castor seed endosperm: evidence from kinetics of inactivation by tetranitromethane

Inactivation of isocitrate lyase (native and EDTA-dialysed) by excess tetranitromethane (TNM) exhibits, biphasic kinetics, in which half of the initial activity is lost in a fast and the remaining half in a slow phase each following the pseudo-first order kinetics. Rate constants of the two phases are proportional to the TNM concentration. High succinate concentration protects the enzyme against TNM inactivation only in the slow phase without any effect on the fast phase. With the EDTA-dialysed enzyme, no such protection (against inactivation by TNM) is observed in the presence of succinate or Mg2+ ions. Addition of both these ligands together brings about protection against the slow phase (as with the native enzyme). It has been proposed that the site-site heterogeneity of isocitrate lyase is a consequence of its quaternary structure constraints.     

Irreversible inhibition of bovine lung angiotensin I-converting enzyme with p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and chlorambucyl L-proline and with evidence that an active site carboxyl group is labeled

Bovine lung angiotensin I-converting enzyme is rapidly and irreversibly inactivated by p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and by the chlorambucil derivative of L-proline (chlorambucyl-proline). Chlorambucil is a nitrogen mustard alkylating agent that is used as an antineoplastic drug. At any one concentration, the inactivation is pseudo-first order with time. Inhibition by both substances is active site directed as suggested by the formation of a reversible enzyme-inhibitor complex prior to the alkylation reaction and by the fact that L-Phe-L-Pro, a reversible inhibitor which is competitive with substrate, is also competitive with both irreversible inhibitors in protecting the enzyme against inactivation. The second order rate constant for inactivation increases in the pH range 5-8 and reaches a value of 3.5 X 10(3) M-1 . min-1 for chlorambucil and 4.8 X 10(2) M-1 . min-1 for chlorambucyl-proline. Chlorambucyl [U-14C]L-proline reacts 1:1 with the converting enzyme and the uptake of radioactivity paralleled the loss of enzyme activity with and without protection by Phe-Pro. Once bound, the radioactive chlorambucyl proline was released (as the dihydroxy derivative) by hydroxide ion with a second order rate constant of 2.2 M-1 . min-1 at 25 degrees C. The radioactive label is also removed by hydroxylamine at pH 10. The lability of the irreversibly bound inhibitor in alkali and in hydroxylamine indicates that an ester bond is formed by the alkylation of an aspartic acid or glutamic acid side chain.     

Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.     

Essential residues in angiotensin converting enzyme: modification with 1-fluoro-2,4-dinitrobenzene

The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.     

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.     

The role of arginyl residues in porphyrin binding to ferrochelatase

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.     

Conduritol aziridine: a new mechanism-based glucosidase inactivator

A new mechanism-based glucosidase inactivator, conduritol aziridine (1,2-dideoxy-1,2-epimino-myo-inositol), has been synthesised from myo-inositol. This aziridine inactivates both the beta-glucosidase from Alcaligenes faecalis and the alpha-glucosidase from yeast according to the expected pseudo-first order kinetics. Inactivation constants measured are Ki = 3.0mM, ki = 0.077 min-1 for the beta-glucosidase, and Ki = 9.5mM, ki = 0.39 min-1 for the alpha-glucosidase. Evidence for irreversible inactivation is provided by the lack of reactivation upon dilution of inactivated enzyme into buffer containing substrate.     

Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli

Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.     

Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase

Trinitrobenzene sulfonate rapidly inactivates ribulosebisphosphate carboxylase/oxygenase from both spinach and Rhodospirillum rubrum. With large molar excesses of the reagent, the reactions obey pseudo-first order kinetics and the rates of inactivations are directly proportional to the concentrations of trinitrobenzene sulfonate; thus, there is no indication of reversible complexation of reagent with enzyme. Saturating levels of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduce the rates of inactivations but do not prevent them, thereby suggesting that the groups subject to arylation remain accessible in the enzyme complexed with competitive inhibitor. Characterization of tryptic digests of the inactivated enzymes reveals that Lys-166 of the R. rubrum enzyme and Lys-334 of the spinach enzyme are the only major sites of arylation. Both of these lysines have been assigned to the catalytic site by prior affinity labeling studies and are found within highly conserved regions of primary structure. As a monoanion over a wide pH range, trinitrobenzene sulfonate, for which the carboxylase lacks high affinity, can thus be used to determine the pKa values of the two active-site lysyl epsilon-amino groups. Based on the pH dependency of inactivation of the R. rubrum enzyme by trinitrobenzene sulfonate, the epsilon-amino group of Lys-166 exhibits a pKa of 7.9 and an intrinsic reactivity (ko) of 670 M-1 min-1. In analogous experiments, Lys-334 of the spinach enzyme exhibits a pKa of 9.0 and a ko of 4500 M-1 min-1. Under deactivation conditions (i.e. in the absence of CO2 and Mg2+), the pKa of Lys-334 becomes 9.8 and the ko is increased to 26,000 M-1 min-1. By comparison, the reaction of trinitrobenzene sulfonate with N-alpha-acetyl-lysine reveals a pKa of 10.8 and a ko of 1250 M-1 min-1. The spinach carboxylase, catalytically inactive as a consequence of selective arylation of Lys-334, still exhibits tight binding of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate. Therefore, Lys-334 is not required for substrate binding and may serve a role in catalysis. The unusually low pKa of Lys-166 argues that this residue is also important to catalysis rather than substrate binding.     

Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine.

D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus. In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics. The limiting rate constant for inactivation of the B. sphaericus enzyme is 1.9 min-1, for the B. subilis enzyme it is 0.36 min-1. The number of catalytic events before inactivation is about 450 for the B. sphaericus enzyme and about 800 for the B. subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter. Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles. Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine.     

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.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

Anion transport in red blood cells and arginine-specific reagents. Interaction between the substrate-binding site and the binding site of arginine-specific reagents

Phenylglyoxal is found to be a potent inhibitor of sulfate equilibrium exchange across the red blood cell membrane at both pH 7.4 and 8.0. The inactivation exhibits pseudo-first-order kinetics with a reaction order close to one at both pH 7.4 and 8. The rate constant of inactivation at 37 degrees C was found to be 0.12 min-1 at pH 7.4 and 0.19 min-1 at pH 8.0. Saturation kinetics are observed if the pseudo-first order rate constant of inhibition is measured as a function of phenylglyoxal concentration. Sulfate ions as well as chloride ions markedly decrease the rate of inactivation by phenylglyoxal at pH 7.4, suggesting that the modification occurs at or near to the binding site for chloride and sulfate. The decrease of the rate of inactivation produced at pH 8.0 by chloride ions is much higher than that produced by sulfate ions. Kinetic analysis of the protection experiments showed that the loaded transport site is unable to react with phenylglyoxal. From the data it is concluded that the modified amino acid(s) residues, presumably arginine, is (are) important for the binding of the substrate anion.