Olsen D B, Hepburn T W, Moos M, Mariano P S, Dunaway-Mariano D
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Biochemistry. 1988 Mar 22;27(6):2229-34. doi: 10.1021/bi00406a063.
Reaction of Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) with phosphonoacetaldehyde or acetaldehyde in the presence of NaBH4 resulted in complete loss of enzymatic activity. Treatment of phosphonatase with NaBH4 in the absence of substrate or product had no effect on catalysis. Inactivation of phosphonatase with [3H]NaBH4 and phosphonoacetaldehyde, NaBH4 and [14C]acetaldehyde, or NaBH4 and [2-3H]phosphonoacetaldehyde produced in each instance radiolabeled enzyme. The nature of the covalent modification was investigated by digesting the radiolabeled enzyme preparations with trypsin and by separating the tryptic peptides with HPLC. Analysis of the peptide fractions revealed that incorporation of the 3H- or 14C-radiolabel into the protein was reasonably selective for an amino acid residue found in a peptide fragment observed in each of the three trypsin digests. Sequence analysis of the 3H-labeled peptide fragment isolated from the digest of the [2-3H]phosphonoacetaldehyde/NaBH4-treated enzyme identified N epsilon-ethyllysine as the radiolabeled amino acid. The ability of the phosphonatase competitive inhibitor (Ki = 230 +/- 20 microM) acetonylphosphonate to protect the enzyme from phosphonoacetaldehyde/NaBH4-induced inactivation suggested that the reactive lysine residue is located in the enzyme active site. Comparison of the relative effectiveness of phosphonoacetaldehyde and acetaldehyde as phosphonatase inactivators showed that the N-ethyllysine imine that is reduced by the NaBH4 is derived from the corresponding N-(phosphonoethyl) imine. On the basis of these findings, a catalytic mechanism for for phosphonatase is proposed in which phosphonoacetaldehyde is activated for P-C bond cleavage by formation of a Schiff base with an active-site lysine. Accordingly, an N-ethyllsysine enamine rather than the high-energy acetaldehyde enolate anion is displaced from the phosphorus.
在NaBH₄存在的情况下，蜡状芽孢杆菌膦酰乙醛水解酶（膦酸酶）与膦酰乙醛或乙醛反应会导致酶活性完全丧失。在没有底物或产物的情况下用NaBH₄处理膦酸酶对催化作用没有影响。用[³H]NaBH₄与膦酰乙醛、NaBH₄与[¹⁴C]乙醛或NaBH₄与[2-³H]膦酰乙醛使膦酸酶失活，在每种情况下都会产生放射性标记的酶。通过用胰蛋白酶消化放射性标记的酶制剂并用HPLC分离胰蛋白酶肽段来研究共价修饰的性质。对肽段部分的分析表明，³H或¹⁴C放射性标记掺入蛋白质中对在三种胰蛋白酶消化物中均观察到的一个肽片段中发现的氨基酸残基具有合理的选择性。对从[2-³H]膦酰乙醛/NaBH₄处理的酶的消化物中分离出的³H标记肽段进行序列分析，确定N-ε-乙基赖氨酸为放射性标记的氨基酸。膦酸酶竞争性抑制剂（Ki = 230±20μM）丙酮基膦酸保护酶免受膦酰乙醛/NaBH₄诱导的失活的能力表明，反应性赖氨酸残基位于酶活性位点。膦酰乙醛和乙醛作为膦酸酶失活剂的相对有效性比较表明，被NaBH₄还原的N-乙基赖氨酸亚胺源自相应的N-（膦酰乙基）亚胺。基于这些发现，提出了膦酸酶的催化机制，其中膦酰乙醛通过与活性位点赖氨酸形成席夫碱而被激活以进行P-C键裂解。因此，N-乙基赖氨酸烯胺而不是高能乙醛烯醇化物阴离子从磷上被取代。
