Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.

The contribution of a conformationally mobile, active site loop to the reaction catalyzed by glutamate semialdehyde aminomutase.

The behavior of glutamate semialdehyde aminomutase, the enzyme that produces 4-aminolevulinate for tetrapyrrole synthesis in plants and bacteria, is markedly affected by the extent to which the central intermediate in the reaction, 4,5-diaminovalerate, is allowed to dissociate. The kinetic properties of the wild-type enzyme are compared with those of a mutant form in which a flexible loop, that reversibly plugs the entrance to the active site, has been deleted by site-directed mutagenesis. The deletion has three effects. The dissociation constant for diaminovalerate is increased approximately 100-fold. The catalytic efficiency of the enzyme, measured as k(cat)/K(m) in the presence of saturating concentrations of diaminovalerate, is lowered 30-fold to 2.1 mM(-1) s(-1). During the course of the reaction, which begins with the enzyme in its pyridoxamine form, the mutant enzyme undergoes absorbance changes not seen with the wild-type enzyme under the same conditions. These are proposed to be due to abortive complex formation between the pyridoxal form of the enzyme (formed by dissociation of diaminovalerate) and glutamate semialdehyde itself.

The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis.

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.

Site-directed mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase.

The 3340-bp fragment containing the Escherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5'-3', and 3'-5'). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on the glyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, the E. coli strain GS245 used to express recombinant serine hydroxymethyltransferase was made recA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5'-phosphate binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions of L- and D-alanine confirmed the suggestion that the active site lysine is not the base that removes the alpha-proton from the substrate.

The function of arginine 363 as the substrate carboxyl-binding site in Escherichia coli serine hydroxymethyltransferase.

Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues. Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme-bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs. The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild-type enzyme. The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild-type enzyme and a 15-fold reduction in affinity for glycine and serine. The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate. Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro-aldol cleavage of serine ethyl ester and serinamide. The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild-type enzyme activity with serine, respectively. The R363A mutant enzyme catalyzed the half transamination of D-alanine methyl ester and L-alanine methyl ester at rates similar to the rates of transamination of D-alanine and L-alanine by the wild-type enzyme. The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase. Evidence is also provided that R363 may play a role in the substrate-induced open to closed conformational change of the active site.

Function of the active-site lysine in Escherichia coli serine hydroxymethyltransferase.

Serine hydroxymethyltransferase has a conserved lysine residue (Lys-229) that forms the internal aldimine with pyridoxal 5'-phosphate. In other pyridoxal 5'-phosphate enzymes investigated so far, this conserved lysine residue also plays a catalytic role as a base that removes the alpha-proton from the amino acid substrate. Three mutant forms of Escherichia coli serine hydroxymethyltransferase (K229Q, K229R, and K229H) were constructed, expressed, and purified. The absorbance spectra, rapid reaction kinetics, and thermal denaturation of the mutant analogs were studied. Only the K229Q mutant serine hydroxymethyltransferase resembled the wild-type enzyme. The results indicate that Lys-229 plays a critical role in expelling the product by converting the external aldimine to an internal aldimine. In the absence of Lys-229, ammonia can also catalyze the same function at a much slower rate. However, Lys-229 apparently is not the base that removes the alpha-proton from the amino acid substrate. The K229Q mutant enzyme could catalyze one turnover of either serine to glycine or glycine to serine at rates approaching those of the wild-type enzyme. After one turnover, the mutant enzyme could not expel the product and bind new substrate. The K229Q mutant enzyme can also transaminate D-alanine, which, like the hydroxymethyltransferase activity, also requires removing the alpha-proton from the substrate. The absorbance spectra of the K229R and K229H serine hydroxymethyltransferases showed that their pyridoxal 5'-phosphate could not readily form an external aldimine with substrates, suggesting that Lys-229 in the wild-type enzyme may never bear a positive charge, further evidence that it is not the base that removes the alpha-proton.

Serine hydroxymethyltransferase: origin of substrate specificity.

All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate. For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue. The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties. The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme. The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme. The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme. The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme. The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm. Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process. This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of sequence features in internal regions of proteins.

Determination of amino acid replacements or assessment of sequence features localized in internal regions of natural or genetically engineered proteins can be performed in some cases with the expenditure of a minimum amount of work and protein material. The procedure requires fragmentation of a protein sample by a chemical or enzymatic method: one of the fragments should include the sequence tract under investigation, suitably preceded by one or more prolyl residues. Classical Edman degradation can be performed on the whole mixture of fragments, thus avoiding the rate-limiting step of peptide purification. Sequence data become readable after performance of reaction with o-phthalaldehyde at the level of proline residues in the relevant peptide. Two specific cases illustrate the potentiality of the procedure.

The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase.

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain.

The primary structure of rabbit liver cytosolic serine hydroxymethyltransferase.

The complete amino acid sequence of cytosolic serine hydroxymethyltransferase from rabbit liver was determined. The sequence was determined from analysis of peptides isolated from tryptic and cyanogen bromide cleavages of the enzyme. Special procedures were used to isolate and sequence the C-terminal and blocked N-terminal peptides. Each of the four identical subunits of the enzyme consists of 483 residues. The sequence could be easily aligned with the sequence of Escherichia coli serine hydroxymethyltransferase. The primary structural homology between the rabbit and E. coli enzymes is about 42%. The importance of the primary and predicted secondary structural homology between the two enzymes is discussed.

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart, and inferences on evolution of the isoenzymes.

We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme from Escherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the gene-duplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10(9) years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme from E. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3 X 10(9) years ago.

The primary structure of mitochondrial aspartate aminotransferase from human heart.

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.

Serine hydroxymethyltransferase from Escherichia coli: purification and properties.

Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M. Plamann, L. Stauffer, M. Urbanowski, and G. Stauffer, Nucleic Acids Res. 11:2065-2074, 1983). The enzyme contained no disulfide bonds but had one sulfhydryl group on the surface of the protein. Several sulfhydryl reagents reacted with this exposed group and inactivated the enzyme. Spectra of the enzyme in the presence of substrates and substrate analogs showed that the enzyme formed the same complexes and in similar relative concentrations as previously observed with the cytosolic and mitochondrial rabbit liver isoenzymes. Kinetic studies with substrates showed that the affinity and synergistic binding of the amino acid and folate substrates were similar to those obtained with the rabbit liver isoenzymes. The enzyme catalyzed the cleavage of threonine, allothreonine, and 3-phenylserine to glycine and the corresponding aldehyde in the absence of tetrahydrofolate. The enzyme was also inactivated by D-alanine caused by the transamination of the active site pyridoxal phosphate to pyridoxamine phosphate. This substrate specificity was also observed with the rabbit liver isoenzymes. We conclude that the reaction mechanism and the active site structure of E. coli serine hydroxymethyltransferase are very similar to the mechanism and structure of the rabbit liver isoenzymes.

Partial amino-acid sequence and cysteine reactivities of cytosolic aspartate aminotransferase from horse heart.

Cytosolic aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from horse heart has five cysteine residues, two of which can be titrated with 5,5'-dithiobis(2-nitrobenzoid acid) in the native enzyme with no impairment of catalytic activity. The rate of modification is unaffected by the presence of substrates. Reaction with N-ethylmaleimide leads to loss of catalytic activity, the rate of inactivation being increased by the presence of substrates. Peptides containing 361 amino-acid residues (about 88% of the total number in the protein) have been isolated and aligned by comparison with the known sequence of the isotopic isoenzyme from pig heart. In the regions compared, 342 of the residues are identical. Hence, assuming that those regions are representative of the whole, then the cytosolic isoenzymes from horse and from pig have about 95% identity of structure. Uniquely among the mammalian cytosolic aspartate aminotransferases so far examined, the enzyme from horse heart is acetylated at the N-terminus.

Sequence homology between prokaryotic and eukaryotic forms of serine hydroxymethyltransferase.

The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins.

Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes.

Sulphydryl groups of mitochondrial aspartate aminotransferase from horse heart were titrated with 5,5'-dithiobis (2-nitrobenzoic acid). From analysis of peptic peptides, 378 amino acid residues (94.3% of the total) in the protein were identified. The results of amino acid sequence analysis are compared with those of cytosolic and mitochondrial aspartate aminotransferases from other sources.