Alter G M, Casazza J P, Zhi W, Nemeth P, Srere P A, Evans C T
Pre-Clinical Science Unit, Department of Veterans Affairs Medical Center, Dallas, Texas 75216.
Biochemistry. 1990 Aug 21;29(33):7557-63. doi: 10.1021/bi00485a003.
Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375. The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity. Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum. No activity was detected for Asn375 or Gln375. The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity. The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs. A mechanism is proposed that electrostatically links His274 and Asp375.
在猪柠檬酸合酶（PCS）的活性位点中，两个氨基酸残基His274和Asp375分别被Gly274、Arg274、Gly375、Asn375、Glu375和Gln375取代。非突变蛋白和突变蛋白在大肠杆菌中表达并纯化，通过初速度和全程动力学分析、亲和柱层析行为以及单克隆抗体反应性，研究了这些氨基酸取代对PCS蛋白整体反应速率和构象的影响。以类似方式纯化的天然蛋白和突变蛋白的亚基分子量为50,000，用10种独立的α-PCS单克隆IgG或多克隆抗PHCS血清检测时具有同源性。未检测到Asn375或Gln375的活性。然而，与非突变酶活性相比，其他纯化的突变蛋白的催化常数（kcats）降低了约10³。在Glu375蛋白中，草酰乙酸的米氏常数（Km）降低了10倍，在Gly274和Arg274 PCS中降低了一半，而在Gly274、Arg274和Gln375 PCS中，乙酰辅酶A的Km降低了2至3倍。提出了一种将His274和Asp375通过静电作用联系起来的机制。
