# Kinetic and Thermodynamic Characterization of Human 4-Oxo-l-proline Reductase Catalysis

## TL;DR

This paper studies the human enzyme BDH2, revealing its role in reducing 4-oxo-l-proline to an anticancer compound and detailing its catalytic and thermodynamic properties.

## Contribution

The paper provides the first mechanistic and thermodynamic characterization of human BDH2's catalytic activity.

## Key findings

- The BDH2 reaction strongly favors forming cis-4-hydroxy-l-proline.
- NADH binding and dissociation rates were measured at different temperatures.
- The reaction's rate is not limited by the chemical step but may involve product release.

## Abstract

The enzyme 4-oxo-l-proline reductase (BDH2)
has recently
been identified in humans. BDH2, previously thought to be a cytosolic
(R)-3-hydroxybutyrate dehydrogenase, actually catalyzes
the NADH-dependent reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline, a compound with known
anticancer activity. Here we provide an initial mechanistic characterization
of the BDH2-catalyzed reaction. Haldane relationships show the reaction
equilibrium strongly favors the formation of cis-4-hydroxy-l-proline. Stereospecific deuteration of NADH C4 coupled with
mass spectrometry analysis of the reaction established that the pro-S hydrogen is transferred. NADH is co-purified with the
enzyme, and a binding kinetics competition assays with NAD+ defined dissociation rate constants for NADH of 0.13 s–1 at 5 °C and 7.2 s–1 at 25 °C. Isothermal
titration calorimetry at 25 °C defined equilibrium dissociation
constants of 0.48 and 29 μM for the BDH2:NADH and BDH2:NAD+ complexes, respectively. Differential scanning fluorimetry
showed BDH2 is highly thermostabilized by NADH and NAD+. The kcat/KM pH–rate profile indicates that a group with a pKa of 7.3 and possibly another with a pKa of 8.7 must be deprotonated and protonated, respectively,
for maximum binding of 4-oxo-l-proline and/or catalysis,
while the kcat profile is largely insensitive
to pH in the pH range used. The single-turnover rate constant is only
2-fold higher than kcat. This agrees with
a pre-steady-state burst of substrate consumption, suggesting that
a step after chemistry, possibly product release, contributes to limit kcat. A modest solvent viscosity effect on kcat indicates that this step is only partially
diffusional. Taken together, these data suggest chemistry does not
limit the reaction rate but may contribute to it.