Acetate and ATP binding in acetyl-CoA synthetase

描述（由申请人提供）：amp形成乙酰辅酶a合成酶（AMP-ACS）是醋酸转化为乙酰辅酶a的主要酶，是合成代谢和分解代谢途径的重要中间体。尽管对AMP-ACS的生理作用的认识取得了进展，但其生物化学方面的关注却很少。AMP-ACS是多种酰基腺苷酸形成酶超家族的成员，该超家族包括酰基辅酶a连接酶、介导肽和聚酮次级代谢产物合成的酶、萤火虫荧光素酶和氨基己二酸还原酶。虽然这个家族的几个成员的结构已经解决了，但AMP-ACS的亲缘关系太远，无法进行分子建模。肠道沙门氏菌AMP-ACS的最新结构现在允许对底物结合和催化的重要活性位点和残基进行预测。提出的研究的两个具体目的是(1)鉴定乙酸结合口袋中的残基(2)鉴定ATP结合的重要残基。通过对肠链球菌AMP-ACSs与丙酰辅酶a合成酶（利用丙酸代替醋酸盐作为底物）和人类SA和MACS 1（分别利用异丁酸盐和辛酸盐）密切相关的结构和序列比对分析，可以鉴定出可能对结合醋酸盐或ATP很重要的候选残基。通过位点定向诱变改变这些残基，然后对所产生的变异进行动力学和生化表征，将描述它们在底物结合中的作用。

DESCRIPTION (provided by applicant): AMP-forming acetyl-CoA synthetase (AMP-ACS) is the principal enzyme for the conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Although progress has been made in understanding the physiological roles of AMP-ACS, its biochemistry has received little attention. AMP-ACS is a member of a diverse superfamily of acyl-adenylate-forming enzymes that includes acyl-CoA ligases, enzymes that mediate the synthesis of peptide and polyketide secondary metabolites, firefly luciferase, and ( aminoadipate reductase. Although structures have been solved for several members of this family, AMP-ACS is too distantly related to allow molecular modeling. The recent structure of the Salmonella enterica AMP-ACS now allows predictions to be made regarding the active site and residues important for substrate binding and catalysis. The two specific aims of the proposed study are (1) to identify residues in the acetate binding pocket and (2) to identify residues important for ATP binding. Analysis of the S. enterica structure and sequence alignment between AMP-ACSs and the very closely related to the propionyl-CoA synthetases, which utilize propionate as substrate instead of acetate, and human SA and MACS 1, which utilize isobutyrate and octanoate, respectively, allowed candidate residues that may be important in binding acetate or ATP to be identified. Alteration of these residues by site-directed mutagenesis followed by kinetic and biochemical characterization of the resulting variants will delineate their role in substrate binding.