Kurlandsky S B, Hilburger A C, Levy H R
Department of Biology, Syracuse University, New York 13244.
Arch Biochem Biophys. 1988 Jul;264(1):93-102. doi: 10.1016/0003-9861(88)90574-7.
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C. It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+. When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+. A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin. This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means. For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively. The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis. A Ki value of 10.5 mM for NADP+ was derived from inhibition studies. The principal conclusion from these studies is that NAD+ binding to L. mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding. Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzyme's conformation, as reflected in a much higher KD value. This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.
来自肠系膜明串珠菌的葡萄糖 -6-磷酸脱氢酶可被胰蛋白酶、胰凝乳蛋白酶、链霉蛋白酶E、嗜热菌蛋白酶、4.0 M尿素以及加热至49℃灭活。葡萄糖 -6-磷酸、NAD⁺和NADP⁺能不同程度地保护该酶免受所有这些形式的灭活。当这些配体的浓度为各自KD浓度的10倍时，NAD⁺或葡萄糖 -6-磷酸的保护作用显著大于NADP⁺的保护作用。对这些配体在不同浓度下对嗜热菌蛋白酶水解葡萄糖 -6-磷酸脱氢酶的保护作用进行了详细分析。该研究证实了上述结论，并允许计算NAD⁺、NADP⁺和葡萄糖 -6-磷酸的KD值，这些值与通过独立方法测定的值一致。对于NADP⁺，可以得出两个KD值，分别为6.1 μM和8.0 mM，分别与低浓度和高浓度NADP⁺对嗜热菌蛋白酶的保护作用相关。前一个值与KD的其他测定结果一致，而后一个值似乎代表NADP⁺与第二个位点的结合，这会导致催化作用受到抑制。从抑制研究中得出NADP⁺的Ki值为10.5 mM。这些研究的主要结论是，与NADP⁺结合相比，NAD⁺与肠系膜明串珠菌葡萄糖 -6-磷酸脱氢酶的结合会导致该酶发生更大的整体构象变化。据推测，与NADP⁺相比，NAD⁺结合的自由能中相当大的一部分用于改变酶的构象，这反映在更高的KD值上。这可能在使这种双核苷酸特异性脱氢酶能够在同一结合位点容纳NAD⁺或NADP⁺方面发挥重要作用。
