CONVERTING TRYPSIN TO CHYMOTRYPSIN - RESIDUE-172 IS A SUBSTRATE-SPECIFICITY DETERMINANT

被引:139
作者
HEDSTROM, L
PERONA, JJ
RUTTER, WJ
机构
[1] UNIV CALIF SAN FRANCISCO,SCH PHARM,HORMONE RES INST,DEPT PHARMACEUT CHEM,SAN FRANCISCO,CA 94143
[2] UNIV CALIF SAN FRANCISCO,DEPT BIOCHEM & BIOPHYS,SAN FRANCISCO,CA 94143
关键词
D O I
10.1021/bi00195a017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Trypsin and chymotrypsin have very similar tertiary structures, yet very different substrate specificities. Recent site-directed mutagenesis studies have shown that mutation of the residues of the substrate binding pocket of trypsin to the analogous residues of chymotrypsin does not convert trypsin into a protease with chymotrypsin-like specificity. However, chymotrypsin-like substrate specificity is attained when two surface loops are changed to the analogous residues of chymotrypsin, in conjunction with the changes in the S1 binding site [Hedstrom, L., Szilagyi, L., and Rutter, W. J. (1992) Science 255, 1249-1253). This mutant enzyme, Tr-->Ch[S1+L1+L2], is improved to a protease with 2-15% of the activity of chymotrypsin by the mutation of Tyr172 to Trp. Residue 172 interacts synergistically with the residues of the substrate binding pocket and the loops to determine substrate specificity. Further, these trypsin mutants demonstrate that substrate specificity is determined by the rate of catalytic processing rather than by substrate binding.
引用
收藏
页码:8757 / 8763
页数:7
相关论文
共 26 条
[1]  
[Anonymous], 1985, ENZYME STRUCTURE MEC
[2]  
BLOW DM, 1971, ENZYMES, V3, P213
[3]   INHIBITION OF CHYMOTRYPSIN BY PEPTIDYL TRIFLUOROMETHYL KETONES - DETERMINANTS OF SLOW-BINDING KINETICS [J].
BRADY, K ;
ABELES, RH .
BIOCHEMISTRY, 1990, 29 (33) :7608-7617
[4]   LOCATION OF DISULPHIDE BRIDGES BY DIAGONAL PAPER ELECTROPHORESIS - DISULPHIDE BRIDGES OF BOVINE CHYMOTRYPSINOGEN A [J].
BROWN, JR ;
HARTLEY, BS .
BIOCHEMICAL JOURNAL, 1966, 101 (01) :214-&
[5]  
CRAIK CS, 1984, J BIOL CHEM, V259, P4255
[6]   SUBSTRATE-SPECIFICITY OF TRYPSIN INVESTIGATED BY USING A GENETIC SELECTION [J].
EVNIN, LB ;
VASQUEZ, JR ;
CRAIK, CS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (17) :6659-6663
[7]   MECHANISM OF CHYMOTRYPSIN - STRUCTURE, REACTIVITY, AND NONPRODUCTIVE BINDING RELATIONSHIPS [J].
FASTREZ, J ;
FERSHT, AR .
BIOCHEMISTRY, 1973, 12 (06) :1067-1074
[9]   ELECTROSTATIC COMPLEMENTARITY WITHIN THE SUBSTRATE-BINDING POCKET OF TRYPSIN [J].
GRAF, L ;
JANCSO, A ;
SZILAGYI, L ;
HEGYI, G ;
PINTER, K ;
NARAYSZABO, G ;
HEPP, J ;
MEDZIHRADSZKY, K ;
RUTTER, WJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (14) :4961-4965
[10]   CONVERTING TRYPSIN TO CHYMOTRYPSIN - GROUND-STATE BINDING DOES NOT DETERMINE SUBSTRATE-SPECIFICITY [J].
HEDSTROM, L ;
FARRJONES, S ;
KETTNER, CA ;
RUTTER, WJ .
BIOCHEMISTRY, 1994, 33 (29) :8764-8769