Studies on the enzymatic hydrolysis of polyglutamyl folates by chicken liver folyl poly-gamma-glutamyl carboxypeptidase. II. Structural studies.
Further studies on the purified chicken hepatic folyl poly-gamma-glutamyl carboxypeptidase (peptidyl-L-glutamate hydrolase, EC 3.4.12.10) have elucidated some of the structural characteristics of the enzyme. Various analytical studies described reveal 424 amino acid residues in the isolated native enzyme with molecular weight of around 57 900. beta-Mercaptoethanol (14.3 mM) activated the enzyme 2.2-fold and induced reductive cleavage of an interchain disulfide linkage resulting in the splitting of the native enzyme into two active polypeptides (molecular weights 43 000 and 18 000). The constituent polypeptides have identical NH2-terminal residues (valine) and exhibit a high degree of sequence homology as revealed by finger print analyses of their tryptic digests. The 10-fold greater sensitivity of the reductively cleaved enzyme to p-chloromercuribenzoate would imply that active site related sulfhydryl groups are not readily accessible in the native enzyme. Ionic strength effects in the presence of Mn2+ and Na+ and the presence of low urea concentration (0.55 M) result in a further up to 5-fold stimulation of reductively cleaved native enzyme. Citrate inhibited and phosphate induced autolytic degradation of the enzyme. The physiological role of gamma-glutamyl carboxypeptidase has been discussed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D002645:Chickens; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005492:Folic Acid; D005971:Glutamates; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism
D000596:Amino Acids; D002729:Chloromercuribenzoates; D005971:Glutamates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D005492:Folic Acid; D002268:Carboxypeptidases; D011623:gamma-Glutamyl Hydrolase; D014357:Trypsin