Molluscan chymotrypsin-like protease: structure, localization, and substrate specificity.
A messenger RNA encoding a chymotrypsin-like preproprotease is expressed abundantly and specifically in the distal quarter of the intestine of the mollusc Haliotis rufescens (red abalone). Consistent with this finding, a chymotrypsin-like activity was detected at highest concentration in the lumen of this segment of the intestine. Because the complexity of total protein in the distal intestinal fluid was low and the chymotrypsin-like protease was highly expressed, purification of the enzyme to near homogeneity was achieved by a single passage over an anion-exchange resin. The primary specificity of the protease, predicted from homology of the key amino acid residues (Ser189, Gly216, and Ser226) in the substrate binding site with similar residues lining the S1 subsites of other chymotrypsin-like enzymes, was confirmed by hydrolysis of a family of tetrapeptide substrates with different P1 amino acids. The optimal P1 residues include those with bulky, gamma-branched side chains (phenylalanine and leucine) similar to the side chain of the asparagine residue at P1 of the activation peptide of the proenzyme. Thus, unlike zymogens of the pancreatic serine proteases, which are activated by the common tryptic mechanism, the zymogen of the molluscan enzyme appears to be activated by an autocatalytic mechanism, i.e., by cleavage with active chymotrypsin-like protease. Additional unique properties of the enzyme predicted from the primary sequence include an unpaired cysteine, with the potential for internal thiol-disulfide isomerization between two different conformers of the protease.
Groppe
J C
JC
Marine Biotechnology Center, University of California, Santa Barbara 93106.
Morse
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D015870:Gene Expression; D007422:Intestines / Q000201:enzymology; D008969:Molecular Sequence Data; D008974:Mollusca / Q000201:enzymology*; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000032:analysis; D012965:Sodium Chloride; D013379:Substrate Specificity; D014018:Tissue Distribution
D012333:RNA, Messenger; D012965:Sodium Chloride; D004247:DNA; D002918:Chymotrypsin