Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S.

Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 in contrast to nearly all of the other eukaryotic aspartic proteases, which catalyze best in the pH range of 2-4.A possible structural reason for the higher optimal pH of HIV-1 protease is the absence of a hydrogen bond to the carboxyl group of active-site Asp", which is nearly universally present in others.To investigate this hypothesis, we have mutated residue 28 in HIV-1 protease from alanine to serine.Both the wild-type and the mutant A28S enzymes have been overexpressed in Escherichia coli using a chemically synthesized gene and purified for a comparative study in enzyme kinetics.The kcat and K,,, values were determined by a radiometric assay for the wild-type enzyme from pH 3.2 to 7.0, and for the mutant enzyme from pH 3.2 to 6.0.The low pK values of the active site of the free enzyme, pKel, are 3.3 and 3.4 for the wild-type and mutant enzymes, respectively.The low pK values of the active site of the enzyme bound to substrate, pKes,, are 5.1 and 4.3 for the wild-type and mutant enzymes, respectively.The high pK values of the free enzyme, pKe2, are 6.8 and 5.6, and the corresponding ones for the substrate-bound enzyme, pKes2, are 6.9 and 6.0 for the wild-type and mutant enzymes, respectively.The lowering of pK values in mutant HIV-1 protease indicates that the hydroxyl group of Ser2' forms a new hydrogen bond to active-site AspzS to increase its acidity.Human immunodeficiency virus type 1 (HIV-l),' an etiological agent of the acquired immunodeficiency syndromes ( l ) , encodes a 11-kDa protease in the pol open reading frame.This protease is responsible for an autoproteolytic release of the protease from the gag-pol fusion precursor protein and a subsequent processing of the gag and pol proteins to yield