Serine transhydroxymethylase from rabbit liver. Sequence of anonapeptide at the pyridoxal-5'-phosphate-binding site.
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The amino acid sequence of the coenzyme-binding site of serine transhydroxymethylase from rabbit liver has been determined. After reduction with NaBH4 and aminoethylation, a first sample of enzyme was digested with thermolysin and a single phosphopyridoxyl peptide was isolated. A second sample of similarly treated enzyme was digested with chymotrypsin and three phosphopyridoxyl peptides clearly originating from a unique coenzyme-binding site were isolated. Sequence analysis of these peptides indicate the following structure: Val-Val-Thr-Thr-His(Pxy)-Thr-Leu. Sequence homologies of the active site of various pyridoxalphosphate enzymes are discussed in terms of a possible catalytic role and of evolution of this class of proteins. (+info)
Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase.
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Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5'-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 microM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor. (+info)
The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction.
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1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site. (+info)
Cooperative interactions of tetrahydrofolate with purified pig kidney serine transhydroxymethylase and loss of this cooperativity in L1210 tumors and in tissues of mice bearing these tumors.
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Serine transhydroxymethylase (5,10-methylenetetrahydrofolate: glycine hydroxymethyl transferase, EC 2.1.2.1) purified 200-fold from pig kidneys showed cooperative interactions with tetrahydrofolate with a Hill coefficient (n value) of 3.9 and a substrate concentration at 50% of maximum velocity, the S(0.5) value, of 0.5 mM. The enzyme in mouse liver and kidney homogenates also showed cooperative interactions with tetrahydrofolate. However, the enzyme obtained from L1210 solid tumors of mice, and from livers and kidneys of mice inoculated with L1210 cells exhibited hyperbolic saturation kinetics and gave a Michaelis constant, Km, value of 0.5 mM for tetrahydrofolate. The interaction of serine with the enzyme from pig kidney, from tissues of normal or tumor-bearing mice, or from L1210 tumors was hyperbolic with a Km of 0.9 mM. The specific activities of the enzyme in the L1210 tumor and in mouse liver were 10-fold higher than in pig or mouse kidney. There was no significant change in the levels of the enzyme in mouse liver and kidney on inoculation with L1210 cells. These results suggest that a tumor can bring about biochemical changes in tissues that are distal to the tumor. (+info)
Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi.
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A single form of serine hydroxymethyltransferase (SHMT) was detected in epimastigotes of Trypanosoma cruzi, in contrast to the three isoforms of the enzyme characterized from another trypanosomatid, Crithidia fasciculata [Capelluto D.G.S., Hellman U., Cazzulo J.J. & Cannata J.J.B. (1999) Mol. Biochem. Parasitol. 98, 187-201]. The T. cruzi SHMT was found to be highly unstable in crude extracts. In the presence of the cysteine proteinase inhibitors N-alpha-p-tosyl-L-lysine chloromethyl ketone and Ltrans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, however, the enzyme could be purified to homogeneity. Digitonin treatment of intact cells suggested that the enzyme is cytosolic. T. cruzi SHMT presents a monomeric structure shown by the apparent molecular masses of 69 kDa (native) and 55 kDa (subunit) determined by Sephadex G-200 gel filtration and SDS/PAGE, respectively. This is in contrast to the tetrameric SHMTs described in C. fasciculata and other eukaryotes. The enzyme was pyridoxal phosphate-dependent after L-cysteine and hydroxylamine treatments and it was strongly inhibited by the substrate analog folate, which was competitive towards tetrahydrofolate and noncompetitive towards L-serine. Partial sequencing of tryptic internal peptides of the enzyme indicate considerable similarity with other SHMTs, particularly from those of plant origin. (+info)
A change in reaction specificity of sheep liver serine hydroxymethyltransferase. Induction of NADH oxidation upon mutation of His230 to Tyr.
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Both serine hydroxymethyltransferase and aspartate aminotransferase belong to the alpha-class of pyridoxal-5'-phosphate (pyridoxalP)-dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X-ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of serine hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing L-aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and D-mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 microM and 5. 2 x 10-3 s-1, respectively. This oxidation was not observed with either the wild type serine hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver serine hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction. (+info)
His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis.
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The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-5'-phosphate and other residues at the active site such as S203, K257, H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its importance in catalysis and/or maintenance of oligomeric structure of the enzyme. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction. (+info)
Identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, arcobacter butzleri, and A. butzleri-like species based on the glyA gene.
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Currently, the detection and identification of Campylobacter and Arcobacter species remains arduous, largely due to cross-species phenotypic similarities and a relatively narrow spectrum of biochemical reactivity. We have developed a PCR-hybridization strategy, wherein degenerate primers are used to amplify glyA fragments from samples, which are then subjected to species-specific oligodeoxyribonucleotide probe hybridizations, to identify and distinguish between Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, Arcobacter butzleri, and an A. butzleri-like species. Evaluation of this strategy with genomic DNA from different type strains suggests that this approach is both specific and sensitive and thus may be applicable in a diagnostic assay to identify and differentiate these highly related species. (+info)