Structural determination of the essential serine and glycosylation sites of carboxypeptidase P.
Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Ki's of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.
Zieske
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Applied Biosystems, Inc., Foster City, California 94404.
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Chen
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Yuan
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D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000737:chemistry*; D006031:Glycosylation; D007531:Isoflurophate / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D005810:Multigene Family; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011489:Protein Denaturation; D012689:Sequence Homology, Nucleic Acid; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000145:classification; D015842:Serine Proteinase Inhibitors
D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D007531:Isoflurophate; D012694:Serine; C025427:carboxypeptidase P; D002268:Carboxypeptidases; D012697:Serine Endopeptidases