Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II

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 kcat ≈ 1 s–1. We correlate experimental
kinetic data (including the E424H variant, newly characterized in
this work) for various GCPII mutants (kcat = 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.