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本文用荧光滴定、透析平衡和分子筛柱层析等方法对甘油醛-3-磷酸脱氢酶(以下简称为GAPDH)与配体的结合进行了研究. 荧光滴定的实验结果,GAPDH酶肮与εNAD~+结合为负协同性,如果酶的Cys-149巯基经碘代乙酸或四硫硫酸钠修饰后,则不仅酶与εNAD~+结合减弱,并且其结合的负协同性也明显减弱.与此相应,εNAD~+与酶蛋白结合后εA部分的荧光增强也不如未修饰酶那样明显.分子筛柱层析的结果指出,当酶的Cys-149巯基经化学修饰后,酶与ATP的结合能力也大大下降.这些结果都说明,酶活性部位Cys-149巯基不仅和酶与NAD~+的菸酰胺部分的结合有关,它的修饰还直接影响到酶的腺嘌呤结合部位,从而影响到酶与配体结合的协同性质.荧光滴定和透析平衡的结果还表明,温度升高酶与εNAD~+结合的负协同性增强,酶与ATP的结合则由非协同性变为负协同性.
In this paper, the binding of glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as GAPDH) and ligand was studied by fluorescence titration, dialysis equilibria and molecular sieve column chromatography.The experimental results of fluorescence titration, GAPDH enzyme and εNAD ~ + Binding is negative synergistic, if the enzyme Cys-149 thiol by iodoacetic acid or sodium tetrasulfite modified, not only enzyme and epsilonNAD ~ + binding weakened, and its combination of negative synergy is also significantly reduced with this Correspondingly, the fluorescence enhancement of εA part after εNAD ~ + binding to enzyme protein was not as obvious as that of unmodified enzyme.The results of molecular sieve column chromatography indicated that when the enzyme Cys-149 mercapto group was chemically modified, the binding ability of enzyme and ATP Greatly decreased.These results indicate that the active site of Cys-149 mercapto not only binds to the nicotinamide moiety of NAD ~ +, but also affects the enzymatic activity of adenine binding site The results of fluorescence titration and dialysis equilibrium also showed that the negative synergism of temperature rising enzyme binding to εNAD ~ + was enhanced and the binding of enzyme to ATP changed from non-synergistic to negative synergistic.