Mechanistic insights into human 4-oxo-L-proline reductase (BDH2) from pre-steady-state kinetics and isotope effects

The human enzyme BDH2 catalyses the reversible NADH-dependent reduction of 4-oxo-L-proline (4oLP) to cis-4-hydroxy-L-proline (c4hLP). c4hLP was not known to occur in mammals until this discovery; furthermore, this compound has anticancer properties. BDH2 itself is also involved in multiple cancers, playing a role in regulating apoptosis and the cell cycle. Oncological research on BDH2 precedes its functional characterisation, however, and so the mechanism by which BDH2 acts in cancers remains elusive, but could be explored by using an inhibitor as a chemical probe in cancer cells. Rational development of such an inhibitor can be informed with knowledge of the catalytic mechanism of BDH2, to which end we have explored the action of this enzyme via steady- and pre-steady-state kinetics, isotope effect studies, and biophysical assays. We have discovered that BDH2 has a tight binding affinity for NADH, and that c4hLP formation is highly thermodynamically favoured. Interaction between the enzyme and NAD(H) confers significant thermostability to BDH2, which has a very low melting temperature without addition of NAD(H). It is therefore posited that BDH2 effectively exists as NAD(H)-bound in the cell, resulting in a pseudo-ordered reaction mechanism in which substrate binding may not be strictly ordered, but is ordered in practice due to high affinity. Stereospecific deuteration of NADH establishes that the pro-S hydrogen is transferred in the reaction. Kinetic isotope effect studies demonstrate that the chemical step of the reaction is very fast and largely unperturbed by unfavourable reaction conditions such as pH, temperature or use of NADPH for which affinity is low. An ionisable group with a pKₐ of 7.3 must be deprotonated for optimal 4oLP binding and/or catalysis. Steady- and pre-steady-state kinetics show that at least one step post-chemistry is partially rate-limiting; we hypothesise that this is likely departure of NAD⁺ from the active site after catalysis.