Recombinant rat guanidinoacetate methyltransferase: structure and function of the NH2-terminal region as deduced by limited proteolysis.
Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with Mr 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH2-terminal segment after Arg20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg20 is also slowed by cross-linking Cys15 and Cys90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp19, and elastase cleaves the bond after Ala24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19-24 is highly exposed to the solvent and flexible. The results also indicate that the NH2-terminal region is not directly involved in substrate binding but plays a role in catalysis.
Fujioka
M
M
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Takata
Y
Y
Gomi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D050937:Guanidinoacetate N-Methyltransferase; D006868:Hydrolysis; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D014357:Trypsin / Q000494:pharmacology
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase; D010196:Pancreatic Elastase; D014357:Trypsin; C004946:glycocyamine; D005998:Glycine