pH dependent inhibition from ammonium ions in the Pseudomonas mevalonii HMG-CoA Reductase crystallization environment

To understand the factors limiting activity in the HMGR crystal with mevalonate, CoA and NAD, we studied the effect of crystallization components and pH on enzymatic activity. We observed a strong inhibition in the crystallization buffer and an increase in activity with increasing pH. We attribute this inhibitive effect to the presence of ammonium ions present in the crystal since inhibition is also observed with several other ammonium salt buffers. Additionally, the lack of inhibition was observed in the absence of ammonium. The effect of each ligand (mevalonate, CoA and NAD) on the rate of the enzymatic reaction in the crystallization environment was further investigated by measuring their K in the crystallization buffer. The K measurements indicate that the hydride transfer step between NAD and mevalonate is inhibited in the crystallization environment. To test this further, we solved a crystal structure of pmHMGR bound to the post-hydride transfer intermediate (mevaldehyde) and cofactor Coenzyme A. The resulting turnover with the formation of a thiohemiacetal indicated that the crystallization environment inhibited the oxidative acylation of mevalonate and the reaction intermediate mevaldyl-CoA.