Nuclear-membrane-associated protein kinase and substrates. The effect of growth state on activity and specificity.

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.     

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.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

Regulation of skeletal muscle AMP deaminase: lysine residues are critical for the pH-dependent positive homotropic cooperativity behaviour of the rabbit enzyme

Reaction of rabbit skeletal muscle AMP deaminase with a low molar excess of trinitrobenzene sulfonic acid (TNBS) results in conversion of the enzyme into a species with about six trinitrophenylated lysine residues per molecule which no longer manifests positive homotropic cooperativity at pH 7.1 or at the optimal pH value of 6.5 in the presence of low K+ concentrations. Substitution of the reactive thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) does not protect the enzyme from the TNBS-induced changes of the catalytic properties, indicating that cysteine residues modification is not at the basis of the effects of TNBS treatment on AMP deaminase and strongly suggesting the obligatory participation of lysine residues to the constitution of a regulatory anionic site to which AMP must bind to stimulate the enzyme at alkaline pH. The TNBS-treated enzyme is also completely desensitized to inhibition by ATP, but not to inhibition by GTP and stimulation by ADP. This observation suggests a connection between the operation of the hypothesized anionic activating site, responsible for positive homotropic cooperativity, and the inhibition exerted by anionic compounds that compete for the same site, among them the most efficient metabolite being probably ATP.     

分枝犁头霉α-半乳糖苷酶的氨基酸组成及化学修饰

α-半乳糖苷酶进行氨基酸组分分析,结果为含有较多的酸性及巯水性氨基酸,较少的组氨酸、酪氨酸及半胱氨酸。 用几种蛋白质侧链修饰试剂对α-半乳糖苷酶进行化学修饰。在一定条件下,当巯基及酪氨酸残基分别被NEM、IAA及NAI修饰后,酶活力不受影响,说明这些基团与活力无关。当羟基、组氨酸及色氨酸残基分别被EDC、DEP、NBS及HNBB修饰后,酶活力大幅度下降,说明这些基团或者参与了酯催化作用或者位于酯活性位区附近。     

粘质赛氏菌胞外蛋白酶的化学修饰

用九种化学修饰剂研究了粘质赛氏菌ＳｅｒｒａｔｉａＭａｒｃｅｓｃｅｎｓ４１００３（２）胞外蛋白酶分子中氨基酸侧链基团与酶催化活性的关系,结果表明组氨酸、丝氨酸、赖氨酸、精氨酸、谷氨酸及天冬氨酸等残基与酶活性无关;半胱氨酸残基与酶活性也无直接关系;而酪氨酸和色氨酸残基侧链的修饰引起酶活力大幅度下降,说明酪氨酸和色氨酸残基为酶活力必需．     

Clostridium pasteurianum glutamine synthetase mechanism. Evidence for active site tyrosine residues

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo-first-order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both transferase and synthetase activities.     

The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.     

Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa

1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride.     

Essential carboxy groups in xylanase A.

An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.     

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by diethyl pyrocarbonate.

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.     

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.     

Identification of essential tyrosine and lysine residues in angiotensin converting enzyme: evidence for a single active site

Inactivation of rabbit lung angiotensin converting enzyme (ACE) by 1-fluoro-2,4-dinitrobenzene (Dnp-F) has been shown to be due primarily to the modification of a tyrosine residue [Bünning, P., Kleeman, S.G., & Riordan, J.F. (1990) Biochemistry (preceding paper in this issue)]. Rabbit testicular ACE is also inactivated by Dnp-F. The specific site of modification has been identified by peptide mapping of tryptic digests of the Dnp-modified protein. Two principal 340-nm-absorbing peaks, not observed with protein modified in the presence of inhibitor, have been characterized. Amino acid and sequence analyses show that these peptides contain two distinct residues that have been selectively modified. The sequence of the major (greater than 90% of the total) modified peptide is YVEFTNK with the Dnp group on tyrosine. The sequence of the second, minor peptide is KVQDLQR with the Dnp group on lysine. Identical peptides were obtained from Dnp-modified rabbit lung ACE. These modified amino acids correspond to residues 200 and 118, respectively, in testicular ACE (human enzyme numbering). Both peptides are present only in the carboxy-terminal half-domain of lung ACE, corresponding to residues 776 and 694, respectively. These results indicate that the Dnp-F sensitive, catalytically functional active site is located in the "testicular" half of lung ACE.     

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.     

Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.     

The molecular weight and thiol residues of acetyl-coenzyme A synthetase from ox heart mitochondria

1. A constant molecular weight of 57000 was obtained by gel filtration of highly purified acetyl-CoA synthetase over a 1000-fold range of enzyme concentrations. The amino acid analysis is reported. 2. With native enzyme at 20 degrees C the relatively rapid reaction of four thiol residues with p-hydroxymercuribenzoate caused an immediate inhibition reversible by either CoA or mercaptoethanol. Other substrates did not protect against this rapid inhibition. 3. The much slower reaction of the remaining four thiol residues was independent of the concentration of the mercurial, first-order with respect to enzyme, and had a large energy of activation (+136kJ/mol), suggesting that a conformation change in the protein was rate-limiting. This slow phase of the reaction was accompanied by an irreversible inactivation of the enzyme. 4. The effects of substrates on this irreversible inactivation at pH7.0 in 5 mm-MgCl(2) indicated strong binding of ATP and pyrophosphate by the enzyme (concentrations for half-maximal effects, K((1/2)), were <30mum and <10mum respectively) and weaker binding of acetyl-CoA (K((1/2)) about 1 mm), AMP (K((1/2)) about 2mm) and acetate. In the presence of acetate, MgCl(2) and p-hydroxymercuribenzoate, titration of the enzyme with ATP revealed at least two ATP binding sites/mol. 5. The experiments suggest that reaction of the thiol residues with mercurial causes loss of enzymic activity by altering the structure of the enzyme, rather than that the thiol residues play a direct role in the catalysis.     

Modification of mouse testicular lactate dehydrogenase by pyridoxal 5''-phosphate.

1. Mouse C4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.7 and 25 degrees C loses activity gradually; 1mM-pyridoxal 5'-phosphate causes 83% inactivation, and higher concentrations of the reagent cause no further loss of activity. 2. The final extent of inactivation is very pH-dependent, greater inactivation occurring at the high pH values. 3. Inactivation may be fully reversed by addition of cysteine, or made permanent by reducing the enzyme with NaBH4. 4. The absorption spectrum of inactivated reduced enzyme indicates modification of lysine residues. Inactivation by 80% corresponds to modification of at least 1.8 mol of lysine/mol of enzyme subunit. 5. There is no loss of free thiol groups after inactivation with pyridoxal 5'-phosphate and reduction of the enzyme. 6. NAD+ or NADH gives complete protection against inactivation. protection studies with coenzyme fragments indicate that the AMP moiety is largely responsible for the protective effect. Lactate (10 mM) gives no protection in the absence of added nucleotides, but greatly enhances the protection given by ADP-ribose (1 mM). Thus ADP-ribose is able to trigger the binding of lactate. 7. Pyridoxal 5'-phosphate also acts as a non-covalent inhibitor of mouse C4 lactate dehydrogenase. The inhibition is non-competitive with respect to both NAD+ and lactate. 8. Km values for the enzyme at pH 8.0 and 25 degrees C, with the non-varied substrate saturating, are 0.3 mM-lactate and 5 microM-NAD+. 9. These results are discussed and compared with pyridoxal 5'-phosphate modification of other lactate dehydrogenase isoenzymes and related dehydrogenases.     

Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system.

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).     

Specific modification of the GTP binding sites of rat 5'-adenylic acid aminohydrolase by periodate-oxidized GTP.

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC3.5.4.6) can be inactivated by incubation with the periodate-oxidized analogue of the enzyme inhibitor GTP. 2. Nucleoside triphosphates and KCl at high concentrations protect against inactivation, while ADP has no effect. 3. The inactivation can be reversed by the addition of GTP and amino acids and made irreversible by reduction with NaBH4. This indicates that, in the binding of the oxidized GTP to the enzyme, a Schiff base is formed between the aldehyde groups of the inhibitor and amino groups of the enzyme. 4. The kinetic properties of the reduced (oxidized GTP)-AMP deaminase derivative indicate that the loss of activity results from an increase in Km while no appreciable change in V is observed; consequently, the enzyme shows positive homotropic cooperativity even in the presence of optimal KCl concentration. 5. Since the treated enzyme shows kinetic properties similar to those of the native enzyme in the presence of GTP, and since the loss of sensitivity to GTP is directly proportional to the degree of inactivation, it is concluded that the oxidized GTP specifically modifies the binding sites for GTP. 6. Binding of the radioactive oxidized GTP shows that two binding sites for this reagent exist in the AMP deaminase molecule.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.