EFFECT OF POINT MUTATIONS ON THE KINETICS AND THE INHIBITION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE - RELATIONSHIP TO DRUG-RESISTANCE

Mutations of human immunodeficiency virus type 1 (HIV-1) protease at four positions, Val(82), Asp(30), Gly(48), and Lys(45) were analyzed for the resulting effects on kinetics and inhibition. In these mutants, Val(82) was substituted separately by Asn, Glu, Ala, Ser, Asp, and Gln; Asp(30) was individually substituted by Phe or Trp; Gly(48) by His, Asp, and Tyr, respectively; and Lys(45) by Glu. By examination of the inhibition of a single inhibitor, the differences in K-i values between the native and mutant enzymes can range from very large to insignificant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of K-i changes was observed for a group of inhibitors. Thus, how much the inhibition changes from the wild-type enzyme to a mutant is dependent on both the mutation and the inhibitor. The examination of K-i changes of inhibitors with closely related structures binding to Val(82) mutants also reveals that the change of inhibition involves subsites in which Val(82) is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of the mutants, the k(cat) and K-m values of many Val(82) mutants and a Lys(45) mutant are comparable to the native enzyme. Surprisingly, Gly(48) mutations produce enzymes with catalytic efficiency superior to that of the wild-type enzyme by as much as 10-fold. Modeling of the structure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region of the mutants. The examination of the relative changes of inhibition and catalysis of mutants suggests that some of the Val(82) and Gly(48) mutants are potential resistance mutants. However, the resistance is specific with respect to individual inhibitors.