Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




William W-C. Chan


The HIV-1 virus encodes a protease essential for the processing of polyprotein precursors into mature viral proteins. This enzyme is a primary target for drug design against AIDS. Concurrent effects of inhibitors targeted to defined regions of the extended active site were investigated using Yonetani-Theorell kinetics to understand its complex specificity requirements. Kinetic data revealed that the simultaneous presence of two specific inhibitors may increase their binding affinity for the enzyme. A 100-fold enhancement in binding affinity was observed in certain instances. Results from this work showed a correlation between inhibitor synergism and substrate specificity thus implicating subsite interactions in enzyme catalysis.

To facilitate the analysis of enzyme-inhibitor interactions an improved graphical method, the combination plot, was developed as an alternative to the Yonetani-Theorell plot. The method generates a single straight line rather than a family of lines which is the traditional approach in such kinetic studies. The slope of the plot, 1/α, quantitatively measures the extent and nature of interaction between two inhibitors on their target enzyme. The approach was easily extended to analyze, for the first time, the interaction between three competitive inhibitors on an enzyme. The combination approach potentially has broad applications for kinetic analysis.

Combination plots were used in the discovery of gem-dialkyl succinic acid derivatives as a new class of unusually potent reversible inhibitors of carboxypeptidases A and B. 2-Ethyl-2-methylsuccinic acid binds to carboxypeptidases A and B with dissociation constants of 1.1 X 10ˉ⁷ M and 3.4 X 10ˉ⁶ M respectively. The low dissociation constants of the inhibitors for the zinc proteases can be attributed primarily to the gem-dialkyl groups which presumably make very important hydrophobic contacts within the active site. The inhibitors may also act as zinc ligands while possessing sufficient affinity for the carboxyl-recognition site in the enzymes.

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