Two groups of metabolically related enzymes, the Group III family of Fe²⁺-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD⁺ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe²⁺ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.

两组代谢相关的酶，即 Fe ²⁺依赖性乙醇脱氢酶 (ADH) 的第 III 组家族和与激活第 III 组 ADH 的 x (nudix) 水解酶相连的独立的核苷二磷酸亚家族，目前尚未得到充分表征。在这里，我们报告了 III 组 ADH 的稳态初始速度正向（醇 → 醛）反应，即γ-羟基丁酸脱氢酶（GHBDH，UniProt：Q59104），从Cupriavidus necator克隆为融合蛋白。我们还报告了 nudix 水解酶对 GHBDH 反应的影响。在最佳 pH 9.0 下，GHBDH 反应被两种不同的饱和纯化 nudix 水解酶激活约 2 倍，即甲醇芽孢杆菌激活剂 (ACT, UniProt: I3EA59) 和大肠杆菌NudF (UniProt Q93K97) 蛋白。在生理 pH 值约为 7.0 时，ACT 激活>3.5 倍。在 pH 9.0 下，正向未激活和 ACT 激活反应的初始速率表征表明，对于这两种情况，产物琥珀半醛相对于GHB 具有竞争性抑制作用，而其他三种底物-产物组合则具有非竞争性抑制作用。该模式与 Mono-Iso Theorell-Chance 动力学中 NAD ⁺首先结合一致。 GHBDH 中一些可能重要的残基的突变体也得到了表征。 H265 在所有 III 组 ADH 中保守，之前被认为是关键的通用碱基，但它对与 H265A 相关的 GHBDH 活性的帮助仅为约 4 倍。先前提出的四种保守的 Fe ²⁺螯合剂（D193、H197、H261 和 H280）均对 GHBDH 活性至关重要。 提出了对正向亚化学计量 ACT 跨物种刺激和反向反应中确认缺乏 ACT 刺激的两步解释。