3-HYDROXY-3-METHYLGLUTARYL-COENZYME-A REDUCTASE OF HALOFERAX-VOLCANII- ROLE OF HISTIDINE-398 AND ATTENUATION OF ACTIVITY BY INTRODUCTION OF NEGATIVE CHARGE AT POSITION-404

Mutant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases of t he halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG-CoA reductases via charge-charge interaction with the active site histidine. To first verify the sequence-based inferenc e that His 398 is the catalytic histidine of the H. volcanii enzyme, e nzyme H398Q was constructed, purified, and assayed far catalysis of th ree reactions: [1] reductive deacylation of HMG-CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily cat alyzed mevaldehyde reduction. By analogy to hamster HMG-CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms o f the 403-residue H. volcanii enzyme were constructed to model phospho rylation and infer whether attenuated activity involved interaction wi th His 398. Chimeric H. volcanii-hamster enzymes constructed in an eff ort to create an active, phosphorylatable chimeric enzyme were inactiv e or not phosphorylated. We therefore added Asp at position 404 to mim ic the introduction of negative charge that would accompany phosphoryl ation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but cat alyzed reaction [2] at 35% the wild-type rate. These observations are consistent with the model that attenuation of catalytic activity resul ts from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.