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L Schirch

Publications and source records attributed to L Schirch.

16 recordsLinked to original sources
PubMed1983

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.

Amino Acid Sequence↗
PubMed1983

Structure and reactivity of cysteine residues in mitochondrial serine hydroxymethyltransferase.

Six cysteine-containing tryptic peptides were isolated and sequenced from rabbit liver mitochondrial serine hydroxymethyltransferase. 2 of the 6 cysteine residues were located on the surface of the enzyme. These 2 cysteine residues are sufficiently close to each other that they form a disulfide bond when oxidized by periodate. Another of the cysteine residues is exposed upon removal of the active site pyridoxal phosphate. The remaining three cysteines are buried and react with sulfhydryl reagents only when the enzyme is denatured. Blocking of one of the surface sulfhydryls with any of several sulfhydryl reagents results in increased catalytic activity when allothreonine is the substrate but decreased activity when serine and tetrahydrofolate are the substrates. The activation and inhibition effects are on Vmax and not on the affinity of the enzyme for its substrates. Of the six cysteine peptides from the mitochondrial enzyme, three show substantial homology with cysteine-containing peptides from the cytosolic form of the enzyme. For both enzyme forms, one of these homologous pairs is a cysteine residue on the surface of the enzyme. These results suggest that the mitochondrial and cytosolic forms of rabbit liver serine hydroxymethyltransferases are the products of separate genes.

Amino Acid Sequence↗
PubMed1983

Serine hydroxymethyltransferase. 31P nuclear magnetic resonance study of the enzyme-bound pyridoxal 5'-phosphate.

The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serine hydroxymethyltransferase has been investigated by 31P NMR spectroscopy. In the holoenzyme, the pyridoxal-P chemical shift is pH-dependent with a pKa of 6.45. The chemical shift of the bound pyridoxal-P is shifted upfield about 0.3 ppm from the signal for free pyridoxal-P. Saturation of the active site with the substrates L-serine, glycine, and tetrahydrofolate does not alter the chemical shift or the pKa of the phosphate group. The addition of these substrates does, however, alter the absorption and circular dichroism spectra of the bound coenzyme, reflecting environmental changes of the pyridine ring-Schiff's base system. We conclude from these studies that the phosphate group of the bound coenzyme is exposed to the solvent. The reorientation and conformational changes of the pyridoxal-P ring which take place during the formation of enzyme-substrate complexes do not appear to change the environment of the phosphate moiety of the coenzyme.

Animals↗
PubMed1982

Methyl methanethiosulfonate as an active site probe of serine hydroxymethyltransferase.

Using methyl methanethiosulfonate and other sulfhydryl group modification reagents we have studied the structure and function of sulfhydryl groups in rabbit liver cytosolic serine hydroxymethyltransferase. From a tryptic digest of the enzyme, seven cysteine-containing peptides were isolated and sequenced. These peptides contained a total of 8 cysteine residues. There are no disulfide bonds in this enzyme. Of the eight sulfhydryl groups, four react with methyl methanethiosulfonate. Two sulfhydryl groups react rapidly with this reagent without altering enzyme catalytic activity. The remaining two sulfhydryl groups react more slowly and cause loss of greater than 90% of the catalytic activity of the enzyme. This nearly inactive enzyme contains pyridoxal-P and can form an enzyme-substrate complex. However, the complex dissociates from the active site suggesting that one possible role for a sulfhydryl group is to stabilize the enzyme-substrate complex. The sequence of the cysteine-containing peptide which is responsible for the mechanism-based inactivation of serine hydroxymethyltransferase by D-3-fluoroalanine was determined. This sulfhydryl group was shown not to be essential to the enzyme for catalytic activity. Also, the sequence of one of the cysteine peptides shows considerable homology to the active site cysteine peptide from tryptophan synthase.

Amino Acid Sequence↗
PubMed1981

Evidence that tetrahydrofolate does not bind to serine hydroxymethyltransferase with positive homotropic cooperativity.

Previous experiments suggesting that tetrahydrofolate binds to serine hydroxymethyltransferase with positive homotropic cooperativity have been reinvestigated. Our results show that the sigmoid-shaped tetrahydrofolate saturation curve, previously obtained by several other investigators, is due to the instability of tetrahydrofolate in the assay solution. Using a different assay method, we have shown that tetrahydrofolate gives a hyperbolic saturation curve with serine hydroxymethyltransferase. We could find no evidence, as suggested by other investigators, that heating the enzyme during purification destroyed its allosteric properties or that NADH binds to the enzyme as an allosteric effector. Evidence is presented that the loss of tetrahydrofolate during the assay period is due to oxidation by dissolved molecular oxygen.

Animals↗
PubMed1980

Evidence for a sulfhydryl group at the active site of serine transhydroxymethylase.

Iodoacetate reacts rapidly with one sulfhydryl group/subunit on aposerine transhydroxymethylase. The carboxymethylated apoenzyme does not recombine with pyridoxal 5'-phosphate. Under conditions used in the apoenzyme studies, the holoenzyme does not react to an appreciable extent with iodoacetate. The reaction of the apoenzyme with iodoacetate shows pseudo-first order kinetics with a half-life of about 3 min at 0 degrees C and pH 7.0. A pattern of saturation kinetics was found when increasing concentrations of iodoacetate were used. The half-maximum rate of inactivation occurred at 1.5 mM iodoacetate. Phosphate was observed to inhibit competitively the inactivation by iodoacetate with a Ki value of 1.8 mM. No inactivation of aposerine transhydroxymethylase was found when iodoacetamide was used in place of iodoacetate. These experiments suggest that removal of the pyridoxal 5'-phosphate from serine transhydroxymethylase exposes a reactive sulfhydryl group with a nearby cationic center which binds the carboxyl group of iodoacetate. The reactive sulfhydryl group was labeled with [14C]iodoacetate. From a chymotryptic digest, a 14C-containing peptide was isolated and determined to be: His-Pro-Lys-Leu-Ile-Ile-Ala-Gly-Thr-Ser-Cys(Cm)-Tyr.

Binding Sites↗
PubMed1975

Serine transhydroxymethylase. Relaxation and transient kinetic study of the formation and interconversion of the enzyme-glycine complexes.

Serine transhydroxymethylase forms three enzyme-glycine complexes which absorb at 3.43, 425, and 495 nm. Temperature-jump studies show three relaxations. Two of the relaxations are observed at both 343 and 425 nm but not at 495 nm. A slower third relaxation is observed only at 495 nm. The absorbance changes for the two relaxations observable at 343 and 425 nm are in opposite directions suggesting that these relaxations are both attributable to the inter conversion of enzyme speiies absorbing at these two wavelengths. The following mechanism is proposed to explain the relaxation effects. See journal for formula. The forward and reverse rate constants for the first step are 7 X 10-4 M-MINUS1 sminus-1 and 1200 Sminus-1, respectively. The forward and reverse rate constants for the second step are 3200 and 1300 sminus-1, respective.y Stopped flow studies on the rate of formation and breakdown of the complexes absorbing at 343 nm (EX) and 425 nm (EY) are in agreement with the proposed mechanism. Stopped flow studies gave a Kobs of 0.1 Sminus-1 for the formation of the 495-nm absorbing complex. This is compared to the reciprocal relaxation time of 200 Sminus-1 observed in the temperature-jump studies. The addition of tetrahydrofolate to this system increased the Kobs to 275 Sminus-1 in the stopped flow studies and the reciprocal relaxation time to 800 Sminus-1 in the temperature-jump studies. The data do not permit a simple interpretation of the relationship of the 495-nm absorbing complex to those adsorbing at 343 and 425 nm. However, the data do support the interpretation that the ability of tetrahydrofolate to increase by 3 orders of magnitude the enzyme-catalyzed rate of exchange of the alpha-hydrogen of glycine with protons of the solvent is attributable to an increased rate of formation of the 495-nm complex.

Glycine↗