HEPATITIS-A VIRUS 3C PROTEINASE SUBSTRATE-SPECIFICITY

被引:57
作者
JEWELL, DA
SWIETNICKI, W
DUNN, BM
MALCOLM, BA
机构
[1] CHIRON CORP,DIV DRUG DISCOVERY & DEV,4560 HORTON ST,EMERYVILLE,CA 94608
[2] UNIV FLORIDA,J HILLIS MILLER HLTH CTR,DEPT BIOCHEM & MOLEC BIOL,GAINESVILLE,FL 32610
关键词
D O I
10.1021/bi00149a017
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
Hepatitis A virus (HAV) 3C proteinase is responsible for processing the viral precursor polyprotein into mature proteins. The substrate specificity of recombinant hepatitis A 3C proteinase was investigated using a series of synthetic peptides representing putative polyprotein junction sequences. Two peptides, corresponding to the viral polyprotein 2B/2C and 2C/3A junctions, were determined to be cleaved most efficiently by the viral 3C proteinase. The k(cat)/K(m) values determined for the hydrolysis of a further series of 2B/2C peptides, in which C-terminal and N-terminal amino acids were systematically removed, revealed that P4 through P2' amino acids were necessary for efficient substrate cleavage. The substitution of Ala for amino acids in P1 and P4 positions decreased the rate of peptide hydrolysis by 100- and 10-fold, respectively, indicating that the side chains of Gln in P1 and Leu in P4 are important determinants of substrate specificity. Rates of hydrolysis measured for other P1- and P4-substituted peptides indicate that S1 is very specific for the Gln side chain whereas S4 requires only that the amino acid in P4 be hydrophobic. A continuous fluorescence quench assay was developed, allowing the determination of k(cat)/K(m) dependence on pH. The pH rate profile suggests that catalyzed peptide hydrolysis is dependent on deprotonation of a reactive group having a pK(a) of 6.2 (+/-0.2). The results of tests with several proteinase inhibitors indicate that this cysteine proteinase, like other picornaviral 3C proteinases, is not a member of the papain family.
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页码:7862 / 7869
页数:8
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