Serine hydroxymethyltransferase. Effect of proteases on the activity and structure of the cytosolic enzyme.
Homogeneous preparations of cytosolic serine hydroxymethyltransferase from rabbit liver were incubated with several different proteases. Chymotrypsin rapidly cleaves a tetradecapeptide from the NH2-terminal end of the enzyme with the enzyme retaining full catalytic activity. Trypsin digestion results in the release of several small peptides from the NH2-terminal end of the enzyme. The remaining core protein is reduced in molecular mass by about 3500 Da. With L-serine as substrate the core protein has 1.5 times the activity of the native enzyme. The difference in activity is due to a change in Vmax since the Km values for L-serine and tetrahydrofolate are unchanged. When allothreonine is used as the substrate the activity of the trypsin-treated enzyme is unchanged. Ks values for glycine and several folate compounds are also unchanged for the trypsin-digested enzyme. The relative distribution of three glycine-enzyme complexes shows only small differences between the native and trypsin-digested enzyme. Thermal denaturation studies show that the trypsin-digested enzyme has a thermal transition three degrees lower than the native enzyme but the same enthalpy of denaturation. These results suggest that the 25-30 amino acid residues from the NH2-terminal end of the enzyme are not important in determining the catalytic activity and structural stability of the purified enzyme. Several other proteases had no observable effect on the activity and size of the enzyme. All of the proteases tested inactivated the apoenzyme and digested it into small fragments. The loss of enzyme activity in frozen liver is probably the result of the enzyme slowly being converted to the apoenzyme form, which is susceptible to protease degradation.
D000818:Animals; D002384:Catalysis; D002918:Chymotrypsin / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D011817:Rabbits; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D014166:Transferases; D012696:Glycine Hydroxymethyltransferase; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin