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 f our positions, Val(82), Asp(30), Gly(48), and Lys(45) were analyzed fo r 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 betwee n the native and mutant enzymes can range from very large to insignifi cant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of K-i cha nges was observed for a group of inhibitors. Thus, how much the inhibi tion changes from the wild-type enzyme to a mutant is dependent on bot h the mutation and the inhibitor. The examination of K-i changes of in hibitors with closely related structures binding to Val(82) mutants al so reveals that the change of inhibition involves subsites in which Va l(82) is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of t he mutants, the k(cat) and K-m values of many Val(82) mutants and a Ly s(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 struct ure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region o f the mutants. The examination of the relative changes of inhibition a nd catalysis of mutants suggests that some of the Val(82) and Gly(48) mutants are potential resistance mutants. However, the resistance is s pecific with respect to individual inhibitors.