Abstract
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.
Highlights
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
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
Using the DTNB-modifiedenzyme, we found from are notat the active site
Summary
Materials-Glycine, serine, tetrahydrofolate, NADH, NADP, alcohol dehydrogenase, and all sulfhydryl reagents were purchasd from. The concentration of a solution of the holoenzyme in milligrams/milliliter was determined by dividing the absorbance at 280 nm by 0.72. One assay used allothreonine as substrate which is converted by the enzyme to glycine and acetaldehyde. The formation of acetaldehyde was monitored continuously by reducing it with NADH and alcohol dehydrogenase (10). This assay does not require tetrahydrofolate as a co-substrata. The rate of formation of the product methylenetetrahydrofolate was continuously followed by including in the assay solution NADP and methylenetetrahydrofolate dehydrogenase which forms NADPH and methenyltetrahydrofolate (11). After 30 min, additional nonradioactive iodoacetate was added to a final concentration of 50 mM and incubated for an additional 3 h.
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