Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S.
作者： E. Ido , H.P. Han , F.J. Kezdy , J. Tang
DOI: 10.1016/S0021-9258(18)54237-X
关键词:
摘要: Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 contrast to nearly all other eukaryotic aspartic proteases, which catalyze best 2-4. A possible structural reason for higher optimal HIV-1 is absence a hydrogen bond carboxyl group active-site Asp25, universally present others. To investigate this hypothesis, we have mutated residue 28 from alanine serine. Both wild-type and mutant A28S enzymes been overexpressed Escherichia coli using chemically synthesized gene purified comparative study enzyme kinetics. The kcat Km values were determined by radiometric assay 3.2 7.0, 6.0. low pK active site free enzyme, pKe1, are 3.3 3.4 enzymes, respectively. bound substrate, pKes1, 5.1 4.3 high pKe2, 6.8 5.6, corresponding ones substrate-bound pKes2, 6.9 6.0 lowering indicates that hydroxyl Ser28 forms new Asp25 increase its acidity.
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