Critical amino acids in the active site of meprin metalloproteinases for substrate and peptide bond specificity.
The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.
Villa
James P
JP
Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Bertenshaw
Greg P
GP
Bond
Judith S
JS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002766:Cholecystokinin / Q000378:metabolism; D005755:Gastrins / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D055672:Static Electricity; D013379:Substrate Specificity
D005755:Gastrins; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; C013550:gastrin 17; D002766:Cholecystokinin; D008666:Metalloendopeptidases; C053833:meprin A