Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the relative activation energies to 1-2 kcal·mol^-1 accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics. Herein, we employ glutamate carboxypeptidase II (GCPII), a dizinc metallopeptidase, also known as the prostate specific membrane antigen, as a model system. The structure and activity of this major cancer antigen have been thoroughly studied, both experimentally and computationally, which makes it an ideal model system for method development. Its reaction mechanism is quite well understood: the reaction coordinate comprises a "tetrahedral intermediate" and two transition states and experimental activation Gibbs free energy of ∼17.5 kcal·mol^-1 can be inferred for the known k_cat ≈ 1 s^-1. We correlate experimental kinetic data (including the E424H variant, newly characterized in this work) for various GCPII mutants (k_cat = 8.6 × 10^-5 s^-1 to 2.7 s^-1) with the energy profiles calculated by QM/MM and QM-only (cluster model) approaches. We show that the near-quantitative agreement between the experimental values and the calculated activation energies (ΔH^⧧) can be obtained and recommend the combination of the two protocols: QM/MM optimized structures and cluster model (QM) energetics. The trend in relative activation energies is mostly independent of the QM method (DFT functional) used. Last but not least, a satisfactory correlation between experimental and theoretical data allows us to provide qualitative and fairly simple explanations of the observed kinetic effects which are thus based on a rigorous footing.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.