Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin

In addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization. Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.     

A 43-kDa protein of Treponema denticola is essential for dentilisin activity

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.     

Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

Probing the role of Tyr 64 of Treponema denticola cystalysin by site-directed mutagenesis and kinetic studies

Tyr 64, hydrogen-bonded to coenzyme phosphate in Treponema denticola cystalysin, was changed to alanine by site-directed mutagenesis. Spectroscopic and kinetic properties of the Tyr 64 mutant were investigated in an effort to explore the differences in coenzyme structure and kinetic mechanism relative to those of the wild-type enzyme. The wild type displays coenzyme absorbance bands at 418 and 320 nm, previously attributed to ketoenamine and substituted aldamine, respectively. The Tyr 64 mutant exhibits absorption maxima at 412 and 325 nm. However, the fluorescence characteristics of the latter band are consistent with its assignment to the enolimine form of the Schiff base. pK(spec) values of approximately 8.3 and approximately 6.5 were observed in a pH titration of the wild-type and mutant coenzyme absorbances, respectively. Thus, Tyr 64 is probably the residue involved in the nucleophilic attack on C4' of pyridoxal 5'-phosphate (PLP) in the internal aldimine. Although the Tyr 64 mutant exhibits a lower affinity for PLP and lower turnover numbers for alpha,beta-elimination and racemization than the wild type, the pH profiles for their Kd(PLP) and kinetic parameters are very similar. Rapid scanning stopped-flow and chemical quench experiments suggest that, in contrast to the wild type, for which the rate-determining step of alpha,beta-elimination of beta-chloro-L-alanine is the release of pyruvate, the rate-determining step for the mutant in the same reaction is the formation of alpha-aminoacrylate. Altogether, these results provide new insights into the catalytic mechanism of cystalysin and highlight the functional role of Tyr 64.     

Substrate inhibition of L-cysteine alpha,beta-elimination reaction catalyzed by L-cystathionine gamma-lyase of Saccharomyces cerevisiae

The alpha,beta-elimination of L-cysteine catalyzed by Saccharomyces cerevisiae L-cystathionine gamma-lyase (EC 4.4.1.1) was inhibited by the substrate. The absorption spectrum of the holoenzyme in the presence of L-cysteine showed that the substrate inhibition observed in this reaction was due mainly to removal of the cofactor.     

Lysine 263 residue of NPM/B23 is essential for regulating ATP binding and B23 stability

Here, we show that Nucleophsomin/B23 provides lysine 263 as a critical binding site for ATP. Mutagenesis of lysine 263 to asparagine (K263N) disrupts B23 from ATP binding. While B23 WT exclusively localizes to the nucleolus, the B23-K263N is redistributed from the nucleolus to the nucleoplam. Notably, the K263N mutant is unstable, and displayed rapid degradation. Alteration of K263 induced B23 instability through increased ubiquitination and proteaosomal degradation. Moreover, mutation of K263 impedes the mitogenic effect of B23 in PC12 cells. Thus, K263 is a critical site for ATP binding and required for B23 stability, confining B23 in the nucleolus.     

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.     

Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^?1 min^?1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with β subunits complex, the enzyme activity completely disappeared, whereas when β subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.     

Histidine 89 is an essential residue for Hsp70 in the phosphate transfer reaction

Autophosphorylation of Hsp70 is detected in the process of substrate refolding in the presence of adenosine triphosphate (ATP) in the reaction mixture. But to date, the role and mechanism of Hsp70 autophosphorylation have not been elucidated. In this study we determined the site of histidine phosphorylation of Hsp70 as an intermediate in the process of phosphate transfer reaction by site-directed mutagenesis. We selected two possible sites (ie, His89 and His227) of intermediate histidine phosphorylation based on our hypothesis of the transfer of gamma-phosphoryl groups and replacement by glycine and serine. Although an acid labile autophosphorylation intermediate of Hsp70 and its cytidine diphosphate-dependent dephosphorylation were detected in wild-type Hsp70, they were markedly suppressed in the H89S mutation of Hsp70, but not on the H227S mutation. The ATPase activity and ATP synthesis activity of Hsp70 were almost completely suppressed in the H89S and H89G mutations. The role of His89 in the phosphate transfer reaction of Hsp70 is discussed.     

Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5'-diphospho-5'-adenosine and pyridoxal phosphate reactive residue

Pyridoxal 5'-diphospho-5'-adenosine (AP2PL) inhibits lamb kidney (Na,K)-ATPase and that inhibition and covalent modification is blocked by the presence of ATP. After trypsin digestion of the labeled, purified alpha subunit and subsequent peptide mapping of the fluorescently labeled peptides by means of high performance liquid chromatography, the main labeled peptide was further purified and analyzed by amino acid composition analysis and peptide sequencing. The obtained peptide had the sequence Ile470-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys480-Tyr-Gln-Le u-Ser-Ile-His- Lys487. Lysine 480 is the residue modified by AP2PL in the absence, but not in the presence of ATP. The beta subunit is not differentially labeled by AP2PL in the presence or absence of ATP. Interestingly, the same results were obtained using pyridoxal phosphate as the labeling and inactivation reagent, indicating that the specificity of labeling by these reagents is not due to the presence of the adenosine moiety, but instead that the initial recognition of nucleotides by the ATP-binding site of (Na,K)-ATPase may be due to recognition of the phosphate moiety. The amino acid sequence surrounding this lysine residue labeled by both reagents is highly conserved in (Na,K)-ATPase and the related (H,K)-ATPase sequences thus far obtained, which may signify a functional importance for this region of the putative ATP-binding site in these transport proteins.     

Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^–1 min^–1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with subunits complex, the enzyme activity completely disappeared, whereas when subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.     

The dimerization of an alpha/beta-knotted protein is essential for structure and function

alpha/beta-Knotted proteins are an extraordinary example of biological self-assembly; they contain a deep topological trefoil knot formed by the backbone polypeptide chain. Evidence suggests that all are dimeric and function as methyltransferases, and the deep knot forms part of the active site. We investigated the significance of the dimeric structure of the alpha/beta-knot protein, YibK, from Haemophilus influenzae by the design and engineering of monomeric versions of the protein, followed by examination of their structural, functional, stability, and kinetic folding properties. Monomeric forms of YibK display similar characteristics to an intermediate species populated during the formation of the wild-type dimer. However, a notable loss in structure involving disruption to the active site, rendering it incapable of cofactor binding, is observed in monomeric YibK. Thus, dimerization is vital for preservation of the native structure and, therefore, activity of the protein.     

Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates

The anion transporter from human red blood cells, band 3, has been expressed in Xenopus laevis frog oocytes microinjected with mRNA prepared from the cDNA clone. About 10% of the protein is present at the plasma membrane as determined by immunoprecipitation of covalently bound 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) with anti-DIDS antibody. The expressed band 3 transport chloride at a rate comparable to that in erythrocytes. Transport of chloride is inhibited by stilbene disulfonates, niflumic acid, and dipyridamole at concentrations similar to those that inhibit transport in red blood cells: DIDS and 4,4'-dinitro-2,2'-stilbene disulfonate inhibit chloride uptake with Kiapp of 34 nM and 2.5 microM, respectively. Lysine 539 has been tentatively identified as the site of stilbene disulfonate binding. Site-directed mutagenesis of this lysine to five different amino acids has no effect on transport. Inhibition by stilbene disulfonates or their covalent binding was not affected when Lys-539 was substituted by Gln, Pro, Leu, or His. However, substitution by Ala resulted in weaker inhibition and covalent binding. These results indicate that lysine 539 is not part of the anion transport site and that it is not essential for stilbene disulfonate binding and inhibition.     

Evidence for an essential arginyl residue in bovine milk gamma-glutamyltransferase

Treatment of bovine milk gamma-glutamyltransferase with 2,3-butanedione in borate buffer markedly inactivates its gamma-glutamyltransferase activity. Inactivation is prevented by a combination of the gamma-glutamyl donor and acceptor substrates, glutathione, and glycylglycine, but less effectively by only one of them. Serine plus borate of maleate provides no protection against the inactivation. Amino acid analysis of the enzyme treated with butanedione in the presence and absence of the protecting substrate combination indicates that complete inactivation correlates with the modification of a single arginyl residue per molecule. The residue modified is associated with the smaller subunit of the two equal subunits which comprise the enzyme. The butanedione-treated enzyme retains a hydrolytic activity, another but less significant catalytic function of the enzyme. The results indicate that the arginyl residue is involved in recognizing the anionic moiety of the acceptor and in binding it to the acceptor site located on the smaller subunit of the enzyme.     

Evidence for an essential glutamyl residue in yeast hexokinase.

Yeast hexokinase is rapidly inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosyl ethyl ester. Sugar substrates afford a partial protection, which is increased by the addition of ADP. Inactivation of the enzyme takes place concomitantly with the incorporation of 1 mol of nitrotyrosine per mol of 50 000-dalton subunit. Exhaustive proteolytic digestion of the modified protein and isolation of the nitrotyrosyl peptide by affinity chromatography, followed by electrophoresis, lead to the identification of the modified residue as a glutamyl residue. This modification of hexokinase occurs without gross conformational changes. The enzyme still binds its substrates, though binding of the nucleotides is perturbed. While the substrates afford a partial protection, they increase the incorporation of nitrotyrosine ethyl ester into the enzyme. This may be attributed to local conformational changes which their binding induces. It is concluded that a glutamyl residue is essential for yeast hexokinase activity and its catalytic function is discussed.     

Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase.

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.