Jetten M S, Gubler M E, Lee S H, Sinskey A J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Appl Environ Microbiol. 1994 Jul;60(7):2501-7. doi: 10.1128/aem.60.7.2501-2507.1994.
Pyruvate kinase activity is an important element in the flux control of the intermediate metabolism. The purified enzyme from Corynebacterium glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentration at the half-maximum rate (S0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reaction rate was 702 U/mg, which corresponded to an apparent kcat of 2,540 s-1. The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutamicum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of the native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequences of other bacterial pyruvate kinases showed 39 to 44% homology, with some regions being very strongly conserved.
丙酮酸激酶活性是中间代谢通量控制中的一个重要因素。从谷氨酸棒杆菌中纯化得到的该酶，其初始速率对磷酸烯醇丙酮酸浓度呈现出明显的S形依赖性。在负别构效应物ATP存在的情况下，半最大反应速率时的磷酸烯醇丙酮酸浓度（S0.5）从1.2 mM增加到2.8 mM，并且以希尔系数表示的协同性从2.0增加到3.2。AMP则产生相反的效应：S0.5降至0.4 mM，并且该酶几乎不表现出协同性。最大反应速率为702 U/mg，这对应于2540 s-1的表观催化常数。该酶不受1,6-二磷酸果糖的影响，并使用Mn2+或Co2+作为阳离子。谷氨酸棒杆菌pyk基因的序列测定揭示了一个编码475个氨基酸多肽的开放阅读框。根据这些信息以及天然蛋白质的分子量，可以得出丙酮酸激酶是一个236 kDa的四聚体。将推导的多肽序列与其他细菌丙酮酸激酶的序列进行比较，显示出39%至44%的同源性，某些区域高度保守。
