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.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013053:Spectrophotometry; D014166:Transferases / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; C014674:methyl methanethiosulfonate; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D014166:Transferases; D012696:Glycine Hydroxymethyltransferase; D014357:Trypsin