Krautwurst H, Bazaes S, González F D, Jabalquinto A M, Frey P A, Cardemil E
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
Biochemistry. 1998 May 5;37(18):6295-302. doi: 10.1021/bi971515e.
Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.
赖氨酸256是酿酒酵母磷酸烯醇式丙酮酸（PEP）羧激酶保守氨基酸，位于该酶的共有激酶1a序列中，通过定点诱变将其替换为丙氨酸、精氨酸或谷氨酰胺。圆二色性、色氨酸荧光光谱和凝胶排阻色谱表明，这些替换并未导致蛋白质结构发生重大变化。与野生型PEP羧激酶相比，三种变体酶的MnADP Km几乎未改变，但PEP羧化反应的Vmax降低了约20000倍。变体酶的草酰乙酸脱羧酶活性水平与天然蛋白相似；然而，它们缺乏丙酮酸激酶样活性。野生型酿酒酵母PEP羧激酶的酶-MnATP复合物解离常数为1.3±0.3 microM，Lys256Arg、Lys256Gln和Lys256Ala突变体的相应值分别为2.0±0.6 microM、17±2 microM和20±6 microM。这些结果共同表明，带正电荷的残基是MnATP正确结合所必需的，并且赖氨酸256在酿酒酵母PEP羧激酶的磷酸转移过程中对过渡态稳定起着至关重要的作用。
