All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.

• MeSH
• Algorithms MeSH
• Aspartic Acid Endopeptidases chemistry   genetics   metabolism   MeSH
• Point Mutation genetics   MeSH
• Dimerization MeSH
• Fluorescent Dyes metabolism   MeSH
• HIV Protease chemistry   genetics   metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Models, Molecular MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Retroviridae Proteins chemistry   genetics   metabolism   MeSH
• Serine chemistry   genetics   MeSH
• Enzyme Stability genetics   MeSH
• Substrate Specificity MeSH
• Threonine chemistry   genetics   MeSH
• Binding Sites genetics   MeSH
• Hydrogen Bonding MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Endopeptidases MeSH
• Fluorescent Dyes MeSH
• HIV Protease MeSH
• protease p15 MeSH Browser
• Recombinant Proteins MeSH
• Retroviridae Proteins MeSH
• Serine MeSH
• Threonine MeSH

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.

• MeSH
• Endopeptidases genetics   metabolism   MeSH
• HIV Protease genetics   metabolism   MeSH
• Kinetics MeSH
• Hydrogen-Ion Concentration MeSH
• Protein Conformation MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Engineering MeSH
• Retroviridae enzymology   MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Endopeptidases MeSH
• HIV Protease MeSH