CHLOROKETONE HYDROLYSIS BY CHYMOTRYPSIN AND N-METHYLHISTIDYL-57-CHYMOTRYPSIN - IMPLICATIONS FOR THE MECHANISM OF CHYMOTRYPSIN INACTIVATION BY CHLOROKETONES

被引:17
作者
PROROK, M [1 ]
ALBECK, A [1 ]
FOXMAN, BM [1 ]
ABELES, RH [1 ]
机构
[1] BRANDEIS UNIV,GRAD DEPT BIOCHEM,WALTHAM,MA 02254
关键词
D O I
10.1021/bi00198a050
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
We have examined the reaction of N-(benzyloxycarbonyl)-L-alanyl- L-glycyl-L-phenylalanyl chloromethyl ketone (ZAGFCMK) with chymotrypsin (Cht) and have found that, in addition to irreversible alkylation of the enzyme, some of the corresponding hydroxymethyl ketone is produced. For each molecule of hydroxy ketone formed, 3.6 molecules of chymotrypsin are inactivated. Chloroketone hydrolysis is also observed with chymotrypsin methylated at N-3 of the active site histidine (MeCht). The hydrolysis proceeds slowly (k = 0.14 min(-1)). Alkylation of the modified enzyme was not observed. An initial burst of free chloride is detected during the MeCht-catalyzed hydrolysis. The magnitude of the chloride burst is proportional to the enzyme concentration in an approximate 1:1 stoichiometry and indicates a relatively rapid chloride-releasing step which gives rise to an intermediate which is more slowly converted to hydroxy ketone. We have also investigated both the solution and MeCht-mediated hydrolysis of the S isomer of N-acetyl-L-alanyl-L-phenylalanyl chloroethyl ketone (S-AcAFCEK). We have concluded that the nonenzymatic hydrolysis proceeds with inversion of configuration at the stereocenter, while the enzymatic process occurs with retention of configuration. The two nucleophilic displacements attending the MeCht-mediated hydrolysis of S-AcAFCEk imply the formation of an intermediate, possibly of an epoxy ether, formed by internal displacement of the chloride by the oxyanion of the initially generated enzyme-chloroketone hemiketal adduct.
引用
收藏
页码:9784 / 9790
页数:7
相关论文
共 41 条
[1]   A REINVESTIGATION OF MIXED CARBONIC ANHYDRIDE METHOD OF PEPTIDE SYNTHESIS [J].
ANDERSON, GW ;
ZIMMERMAN, JE ;
CALLAHAN, FM .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1967, 89 (19) :5012-+
[2]  
[Anonymous], 1984, SOLID PHASE PEPTIDE, P114
[3]   ELASTASE-CATALYZED HYDROLYSIS OF P-NITROPHENYL TRIMETHYLACETATE [J].
BENDER, ML ;
MARSHALL, TH .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1968, 90 (01) :201-&
[4]   INHIBITION OF CHYMOTRYPSIN BY PEPTIDYL TRIFLUOROMETHYL KETONES - DETERMINANTS OF SLOW-BINDING KINETICS [J].
BRADY, K ;
ABELES, RH .
BIOCHEMISTRY, 1990, 29 (33) :7608-7617
[5]  
BRAYER GD, 1989, P NATL ACAD SCI USA, V76, P96
[6]  
CHEM R, 1979, BIOCHEMISTRY-US, V18, P921
[7]   The relation between the structure of organic halides and the speeds of their reaction with inorganic iodides. III. The influence of unsaturated groups [J].
Conant, JB ;
Kirner, WR ;
Hussey, RE .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1925, 47 :488-501
[8]  
Cromer D T, 1974, INT TABLES XRAY CRYS, VIV, p[99, 149]
[9]  
CROMER DT, 1974, INT TABLES XRAY CRYS, V4, P148
[10]   A FACILE SYNTHESIS OF METHANESULFONATE ESTERS [J].
CROSSLAN.RK ;
SERVIS, KL .
JOURNAL OF ORGANIC CHEMISTRY, 1970, 35 (09) :3195-&