title: Structural basis for elastolytic substrate specificity in rodent alpha-chymases.
abstract: Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases.
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341. Electronic address: jkervine@prdus.jnj.com.
authors.forename: Jukka
authors.initials: J
authors.lastname: Kervinen
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Marta
authors.initials: M
authors.lastname: Abad
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Carl
authors.initials: C
authors.lastname: Crysler
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Michael
authors.initials: M
authors.lastname: Kolpak
authors.affiliation: Research and Early Development, Spring House, Pennsylvania 19477.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mahan
authors.affiliation: Research and Early Development, Spring House, Pennsylvania 19477.
authors.forename: John A
authors.initials: JA
authors.lastname: Masucci
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Shariff
authors.initials: S
authors.lastname: Bayoumy
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Maxwell D
authors.initials: MD
authors.lastname: Cummings
authors.affiliation: Bioinformatics, West Coast Research & Early Development, San Diego, California 92121.
authors.forename: Xiang
authors.initials: X
authors.lastname: Yao
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Matthew
authors.initials: M
authors.lastname: Olson
authors.affiliation: Research and Early Development, Spring House, Pennsylvania 19477.
authors.forename: Lawrence
authors.initials: L
authors.lastname: de Garavilla
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Kuo
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Deckman
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, Structural Biology, Exton, Pennsylvania 19341. Electronic address: jspurli1@prdus.jnj.com.
authors.forename: John
authors.initials: J
authors.lastname: Spurlino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D053818:Chymases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D053818:Chymases