3-DIMENSIONAL STRUCTURE OF CHYMOTRYPSIN INACTIVATED WITH (2S)-N-ACETYL-L-ALANYL-L-PHENYLALANYL ALPHA-CHLOROETHANE - IMPLICATIONS FOR THE MECHANISM OF INACTIVATION OF SERINE PROTEASES BY CHLOROKETONES

被引:22
作者
KREUTTER, K
STEINMETZ, ACU
LIANG, TC
PROROK, M
ABELES, RH
RINGE, D
机构
[1] BRANDEIS UNIV, DEPT BIOCHEM, PROGRAM BIOORGAN CHEM, WALTHAM, MA 02254 USA
[2] BRANDEIS UNIV, DEPT CHEM, PROGRAM BIOPHYS, WALTHAM, MA 02254 USA
[3] BRANDEIS UNIV, ROSENSTIEL BASIC MED SCI RES CTR, WALTHAM, MA 02254 USA
关键词
D O I
10.1021/bi00250a033
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
The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, k(i). The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P-2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P-1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.
引用
收藏
页码:13792 / 13800
页数:9
相关论文
共 49 条
[1]   SYNTHESIS AND PROPERTIES OF PEPTIDYL DERIVATIVES OF ARGINYLFLUOROMETHANES [J].
ANGLIKER, H ;
WIKSTROM, P ;
RAUBER, P ;
STONE, S ;
SHAW, E .
BIOCHEMICAL JOURNAL, 1988, 256 (02) :481-486
[2]   THE SYNTHESIS OF INHIBITORS FOR PROCESSING PROTEINASES AND THEIR ACTION ON THE KEX2 PROTEINASE OF YEAST [J].
ANGLIKER, H ;
WIKSTROM, P ;
SHAW, E ;
BRENNER, C ;
FULLER, RS .
BIOCHEMICAL JOURNAL, 1993, 293 :75-81
[3]  
BANNER DW, 1991, J BIOL CHEM, V266, P20085
[4]   STRUCTURE OF CHYMOTRYPSIN TRIFLUOROMETHYL KETONE INHIBITOR COMPLEXES - COMPARISON OF SLOWLY AND RAPIDLY EQUILIBRATING INHIBITORS [J].
BRADY, K ;
WEI, AZ ;
RINGE, D ;
ABELES, RH .
BIOCHEMISTRY, 1990, 29 (33) :7600-7607
[5]   PH-DEPENDENCE OF THE INHIBITION OF CHYMOTRYPSIN BY A PEPTIDYL TRIFLUOROMETHYL KETONE [J].
BRADY, K ;
LIANG, TC ;
ABELES, RH .
BIOCHEMISTRY, 1989, 28 (23) :9066-9070
[6]   EQUILIBRIUM STUDIES OF WATER AND THIOL ADDITION TO KETONES - SUBSTITUENT AND SOLVENT EFFECTS FOR METHYL KETONES [J].
BURKEY, TJ ;
FAHEY, RC .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1983, 105 (04) :868-871
[7]   AMINO-AROMATIC INTERACTIONS IN PROTEINS [J].
BURLEY, SK ;
PETSKO, GA .
FEBS LETTERS, 1986, 203 (02) :139-143
[8]   REFINED CRYSTAL-STRUCTURE OF GAMMA-CHYMOTRYPSIN AT 1.9 A RESOLUTION - COMPARISON WITH OTHER PANCREATIC SERINE PROTEASES [J].
COHEN, GH ;
SILVERTON, EW ;
DAVIES, DR .
JOURNAL OF MOLECULAR BIOLOGY, 1981, 148 (04) :449-479
[9]  
COLLEN D, 1982, J LAB CLIN MED, V99, P76
[10]   PRELIMINARY X-RAY DIFFRACTION STUDIES OF CRYSTAL FORMS OF FREE AND INHIBITED CHYMOTRYPSIN [J].
COREY, RB ;
BATTFAY, O ;
BRUECKNE.DA ;
MARK, FG .
BIOCHIMICA ET BIOPHYSICA ACTA, 1965, 94 (02) :535-&