Reinhardt Laurie A, Svedruzic Drazenka, Chang Christopher H, Cleland W Wallace, Richards Nigel G J
Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, USA.
J Am Chem Soc. 2003 Feb 5;125(5):1244-52. doi: 10.1021/ja0286977.
Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/- 0.1% and 0.5% +/- 0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/- 0.2% normal. Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/- 0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/- 0.2% normal. The (18)O isotope effect on the formation of CO(2), however, 0.7% +/- 0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate.
草酸脱羧酶（OxDC）催化一种显著的转化反应，其中草酸中的碳 - 碳键被裂解生成二氧化碳和甲酸。与从黑曲霉中分离出的天然OxDC一样，来自枯草芽孢杆菌的重组细菌OxDC在其静止状态下含有Mn（II），并且需要催化性的双氧来发挥活性。尽管这一假设仍有待明确证实，但OxDC催化碳 - 碳键裂解最可能的机制涉及自由基中间体的参与。通过重组野生型枯草芽孢杆菌OxDC的高分辨率晶体结构（Anand等人，《生物化学》，2002年，41卷，7659 - 7669页），对催化机制的研究已奠定了坚实基础。我们现在报告草酸脱羧酶催化草酸脱羧反应的重原子动力学同位素效应测量结果，这似乎是对OxDC所采用机制的首次详细研究。在pH 4.2时，OxDC催化生成甲酸和CO₂的正常¹³C同位素效应分别为1.5%±0.1%和0.5%±0.1%，而对甲酸生成的¹⁸O同位素效应为正常的1.1%±0.2%。同样在pH 5.7时，甲酸和CO₂的生成分别表现出正常的¹³C同位素效应1.9%±0.1%和0.8%±0.1%，对甲酸生成的¹⁸O同位素效应为正常的1.0%±0.2%。然而，在pH 5.7时，对CO₂生成的¹⁸O同位素效应为0.7%±0.2%，是反常的。这些结果与一个多步模型一致，在该模型中，结合的草酸向锰酶进行可逆的、质子偶联的电子转移，生成草酸自由基，该自由基脱羧产生甲酸根自由基阴离子。随后该中间体的还原和质子化生成甲酸。
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