IONIZATION STATES OF THE CATALYTIC RESIDUES IN HIV-1 PROTEASE

Chemical synthesis was used to prepare the HIV-1 protease specifically C-13-labelled in the catalytically essential Asp 25 in each monomer, The NMR chemical shift of the C-13-enriched homodimeric enzyme was mea sured in the presence of the inhibitor pepstatin, a mimic of the tetra hedral intermediate formed in enzyme catalysis, In this complex, the c atalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2,5-6.5, one Asp 25 side chain is prot onated and the other deprotonated, By contrast, in the absence of inhi bitor the two Asp side chains are chemically equivalent and both depro tonated at pH 6, the optimum for enzymatic activity, These direct obse rvations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of imp roved HIV-1 protease inhibitors.