Parast C V, Wong K K, Kozarich J W, Peisach J, Magliozzo R S
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Biochemistry. 1995 May 2;34(17):5712-7. doi: 10.1021/bi00017a002.
Pyruvate formate-lyase (PFL) is a glycyl radical-containing enzyme that catalyzes the reversible, nonoxidative conversion of pyruvate and CoA into acetyl-CoA and formate. The radical is located on the alpha-carbon of glycine 734 and is required for catalysis. Two cysteine residues, C418 and C419, are also essential for catalysis. Mercaptopyruvate, a biologically relevant pyruvate analog, is shown here to be a mechanism-based inactivator of PFL. Upon addition of mercaptopyruvate to active PFL, an EPR spectrum is generated which exhibits components from two sulfur-based radicals. For one of these radicals, a disulfide radical, the hyperfine coupling to a single beta-methylene hydrogen is resolved in features at g = 2.057 and 2.023. The effects of deuterium labeling of the enzyme on the EPR spectrum for this species are consistent with the new radical being on a cysteine residue, probably cysteine 418 or 419. This spectrum is not formed upon addition of the inactivator to mutant enzymes, C418S and C419S, indicating that both active site cysteines are required for formation of the new radicals. The identity of the second species is also ascribed to be a sulfur-based radical on the basis of the EPR feature found at g = 2.01. Our results constitute the first direct evidence of sulfur-based radical formation in an enzyme. A mechanism for formation of the cysteine-based disulfide radical is proposed which requires the participation of the two active site cysteines as well as the glycyl radical.
丙酮酸甲酸裂解酶（PFL）是一种含甘氨酰自由基的酶，它催化丙酮酸和辅酶A可逆的非氧化转化，生成乙酰辅酶A和甲酸。该自由基位于甘氨酸734的α-碳上，是催化反应所必需的。两个半胱氨酸残基C418和C419对催化反应也至关重要。巯基丙酮酸是一种与生物学相关的丙酮酸类似物，在此被证明是PFL的一种基于机制的失活剂。向活性PFL中加入巯基丙酮酸后，会产生一个电子顺磁共振（EPR）光谱，该光谱显示出两个基于硫的自由基的成分。对于其中一个自由基，即二硫自由基，与单个β-亚甲基氢的超精细偶合在g = 2.057和2.023处的特征中得到解析。酶的氘标记对该物种EPR光谱的影响与新自由基位于半胱氨酸残基上一致，可能是半胱氨酸418或419。向突变酶C418S和C419S中加入失活剂后不会形成该光谱，这表明两个活性位点的半胱氨酸都是形成新自由基所必需的。基于在g = 2.01处发现的EPR特征，第二种物种的身份也被归因于一个基于硫的自由基。我们的结果构成了酶中基于硫的自由基形成的首个直接证据。提出了一种基于半胱氨酸的二硫自由基形成机制，该机制需要两个活性位点的半胱氨酸以及甘氨酰自由基的参与。
