Karsten William E, Cook Paul F
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
Biochemistry. 2007 Dec 18;46(50):14578-88. doi: 10.1021/bi701524z. Epub 2007 Nov 21.
The NAD-malic enzyme catalyzes the oxidative decarboxylation of l-malate. Structures of the enzyme indicate that arginine 181 (R181) is within hydrogen bonding distance of the 1-carboxylate of malate in the active site of the enzyme and interacts with the carboxamide side chain of the nicotinamide ring of NADH, but not with NAD+. Data suggested R181 might play a central role in binding and catalysis in malic enzyme, and it was thus changed to lysine and glutamine to probe its potential function. A nearly 100-fold increase in the Km for malate and a 30-fold increase in the Ki for oxalate, an analogue of the enolpyruvate intermediate, in the R181Q and R181K mutants are consistent with a role for R181 in binding substrates. The mutant enzymes also exhibit a >10-fold increase in KiNADH, but only a slight or no change in KNAD, consistent with rotation of the nicotinamide ring into the malate binding site upon reduction of NAD+ to NADH. The activity of the R181Q mutant can be rescued by ammonium ion likely by binding in the pocket vacated by the guanidinium group of R181. Results suggest 2 mol of ammonia bind per mole of active sites with a high-affinity KNH4 of 0.7 +/- 0.1 mM and a low-affinity KNH4 of approximately 420 mM. Occupancy of the high-affinity site, likely by NH4+, results in an increase in the affinity of malate, oxalate, and NADH (with no change in NAD affinity), consistent with the above-proposed roles for R181. The second molecule to bind is likely neutral NH3, and its binding increases V/Et approximately 20-fold. Primary deuterium and 13C isotope effects measured in the absence and presence of ammonium ion suggest R181Q predominantly affects the rate of the reaction by changing the rate of the precatalytic conformational change. The isotope effects do not change upon binding the second mole of ammonia in spite of the 20-fold increase in V/Et. Thus, the R181Q mutant enzyme exists as an equilibrium mixture between active and less active forms, and NH3 stabilizes the more active conformation of the enzyme.
NAD - 苹果酸酶催化L - 苹果酸的氧化脱羧反应。该酶的结构表明，精氨酸181（R181）在酶活性位点与苹果酸的1 - 羧酸盐处于氢键距离内，并与NADH烟酰胺环的羧酰胺侧链相互作用，但不与NAD⁺相互作用。数据表明R181可能在苹果酸酶的结合和催化中起核心作用，因此将其突变为赖氨酸和谷氨酰胺以探究其潜在功能。在R181Q和R181K突变体中，苹果酸的Km增加近100倍，草酸盐（烯醇丙酮酸中间体的类似物）的Ki增加30倍，这与R181在结合底物中的作用一致。突变酶的KiNADH也增加了10倍以上，但KNAD仅略有变化或无变化，这与NAD⁺还原为NADH时烟酰胺环旋转进入苹果酸结合位点一致。R181Q突变体的活性可以通过铵离子挽救，可能是通过结合R181胍基空出的口袋。结果表明，每摩尔活性位点结合2摩尔氨，高亲和力KNH4为0.7±0.1 mM，低亲和力KNH4约为420 mM。高亲和力位点（可能被NH4⁺占据）导致苹果酸、草酸盐和NADH的亲和力增加（NAD亲和力不变），这与上述R181的作用一致。第二个结合的分子可能是中性NH3，其结合使V/Et增加约20倍。在有无铵离子存在下测量的初级氘和¹³C同位素效应表明，R181Q主要通过改变预催化构象变化的速率来影响反应速率。尽管V/Et增加了20倍，但结合第二摩尔氨时同位素效应不变。因此，R181Q突变酶以活性形式和活性较低形式之间的平衡混合物存在，NH3稳定了酶的更具活性的构象。
