Cadet F, Meunier J C
Centre de Biotechnologie Agro-Industrielle, Institut National Agronomique Paris-Grignon, Thiverval-Grignon, France.
Biochem J. 1988 Jul 1;253(1):249-54. doi: 10.1042/bj2530249.
The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.
本文旨在研究景天庚酮糖-1,7-二磷酸酶的一些稳态动力学性质、其pH依赖性以及底物类似物果糖2,6-二磷酸的影响。实验分别以景天庚酮糖1,7-二磷酸和另一种底物果糖1,6-二磷酸进行。两种底物的pK值相同，果糖2,6-二磷酸表现为竞争性抑制剂。这些结果表明，在酶分子上存在一个对景天庚酮糖1,7-二磷酸或果糖1,6-二磷酸而言独一无二的活性位点。Mg2+浓度增加会使最适pH发生偏移。对于果糖-1,6-二磷酸酶，我们认为这种偏移是由于活性中心附近的负电荷被中和所致[Cadet、Meunier和Ferté（1987年），《欧洲生物化学杂志》162卷，393 - 398页]。景天庚酮糖1,7-二磷酸和果糖1,6-二磷酸的游离形式并非该酶的常见底物，Mg2+也不是。但相对于（Mg2+-底物4-）2-复合物的动力学并不支持该复合物是底物这一观点。本文提出了对此差异的一种解释，涉及活性中心附近的负电荷和Mg2+的正电荷。还原态酶的观测Vmax值对于两种底物均为理论Vmax值的65%，但相对于景天庚酮糖1,7-二磷酸的观测Vmax值是相对于果糖1,6-二磷酸的观测Vmax值的3倍。相对于景天庚酮糖1,7-二磷酸的特异性常数（kcat./Km）为1.62×10(6) M-1·s-1，而相对于果糖1,6-二磷酸的特异性常数为5.5×10(4) M-1·s-1，这表明该酶对景天庚酮糖1,7-二磷酸具有较高但并非绝对的特异性。
