DECARBOXYLATION DURING THE FORMATION OF THE FLUORESCENT NAD DERIVATIVE OF D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

During the formation of the fluorescent NAD derivative of rabbit muscle D-glyceral-dehydc-3-phosphate dehydrogenase modified with ~(14)C-1-iodoacetate by ultraviolet irradiation about 1/2 of the total radioactivity is removed as CO_2, when the fluorescent intensity has reached a maximum, With a weak light source, it can be shown that the formation of the fluorescent derivative precedes the decarboxylation. For the Bacillus stearothermophilus enzyme the formation of the fluorescent NAD derivative is an all-sites reaction and is accompanied by the removal of 4 CO_2 molecules showing conclusively that CO_2 has come from the subunits carrying the fluorophore.It is known that the yeast enzyme can be carboxymethylated either at 4, or under carefully controlled conditions, at 2 of the active site SH groups. Experiments with ~(14)C-labelled NAD~ show that both the tetrakis and his carboxymethylated enzymes form 2 molecules of the fluorescent NAD derivative. However, for the biscarboxymethylated enzyme, only on     

NAD~ ANALOGS IN FORMATION OF NEW FLUOROPHORES ON ULTRAVIOLET IRRADIATION WITH D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

A number of NAD~ analogs have been tested in their ability to form fluorescent deriva-tives when UV irradiated with the active site Cys-149 carboxymethylated GAPDH and this hasbeen compared with their properties of acting as hydrogen acceptors and forming the Rackerband. Among the analogs tested, NHD~ , NGD~ , APAD~ and ∈NAD~ give positive results inall the above-mentioned reactions whereas αNAD~ , NMN~ and CPAD~ are all negative.FPAD~ forms a fluorescent derivative on UV irradiation with the carboxymethylated enzymebut is inactive as a hydrogon aceeptor and does not form the Racker band. This is probablydue to thiohemiacetal formation of the pyridine 3-aldehyde of this derivative with the activesite SH group required for both the latter 2 reactions. TPAD~ , although active as a hydrogenacceptor, does not form either a fluorescent derivative or a Racker band. The fact that forthe great majority of the analogs, the property of forming fluorescent derivatives is is parallelwith their hydrogen acceptor activity seems to show that the formation of the fluorescentderivative is indeed at the active site, and hence can be used as an intrinsic probe for thestudy of the conformation of the active site of this enzyme.     

THE COOPERATIVE BEHAVIOR OF YEAST D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AS STUDIED BY THE FORMATION OF THE FLUORESCENT NAD DERIVATIVE

The ultraviolet irradiation of rite yeast D-glyceraldchyde-3-phosphate dehydrogenase carbox-ymethylated at the active site Cys residues, as with the rabbit muscle enzyme, led to the for-mation of a fluorcscent NAD derivative with an emission maximum at 410 mn. Similar resultswere obtained with the enzyme selectively carboxymethylated at only 2 of its 4 active site Cysresidues. The binding of NAD~ to both the carboxymethylated enzymes is non-cooperative oronly weakly negatively cooperative when determined by NAD~ quenching of thc intrinsic proteinfluorescence, However, determinations of the amount of fluorescent NAD derivative formedunder different NAD~ concentrations show that both the earboxymethylated enzymes appearedto bind NAD~ with positive cooperativity as in the ease of the binding of NAD~ to thenative apoenzyme. This seems to suggest that the spatial positioning of the nicotinamidemoity at the active site of the irradiated enzyme resembles more closely that of the nico-tinamide ring in the     

甘油醛-3-磷酸脱氢酶形成荧光NAD衍生物过程中的脱羧

本文证明兔肌甘油醛-3-磷酸脱氢酶,经紫外光照射,形成荧光NAD衍生物的过程中发生脱羧。用[1-~(14)C]碘乙酸示踪此反应当荧光强度达最大值时,大约有一半的总放射性以二氧化碳的形式放出。用弱光源照射,可以观察到荧光衍生物的生成先于脱羧。嗜热脂肪芽孢杆菌酶,荧光NAD衍生物的生成为全位反应,并伴随有四分子二氧化碳放出。表明二氧化碳只能从形成荧光团的亚基上放出。控制实验条件,酵母酶四个活性部位的巯基可以全被羧甲基化,也可以仅有两个被羰甲基化。这两种酵母酶均形成两分子的荧光NAD衍生物,但二羰甲基酵母酶却只能脱去一分子二氧化碳。这一现象可能表示亚基间存在着构象的不对称性。     

酵母羧甲基甘油醛-3-磷酸脱氢酶光照形成NAD荧光衍生物时的协同作用

本文表明与兔肌酶相同,活性部位巯基羧甲基化的酵母甘油醛-3-磷酸脱氢酶在NAD 存在下,经紫外光照,形成一发射峰为410毫微米的荧光衍生物。对于在四个活性部位中只有两个被引入羰甲基的酵母酶,也得到同样的结果。用蛋白内禀荧光淬灭方法测定这两种羧甲基酶,结合NAD~ 均为极微弱的负协同性,或几乎无协同性;但根据荧光衍生物形成的测定,两者却均为正协同性,这可能是由于NAD~ 和酶朊的结合方位在含荧光衍生物的酶中与天然酶中较为接近所致。     

羧甲基甘油醛-3-磷酸脱氢酶与NAD~ 类似物生成荧光物的研究

本文对一系列NAD~ 类似物和活性部位Cys-149羧甲基化的GAPDH一起紫外光照时,形成荧光衍生物的能力与这些类似物作为氢受体的活力,形成Racker band的能力进行了比较。在被测类似物中NAD~ ,NGD~ ,APAD~ 和eNAD~ 对上述三个反应均为正结果;而a-NAD~ ,NMN~ 和CPAD~ 均为负结果,FPAD~ 与羧甲基酶一起紫外光照时能形成荧光衍生物,但无氢受体活力,也不形成Racker band,这可能是由于FPAD的吡啶环3-醛基与活性部位的—SH基团形成了硫半缩醛,而该—SH基团正是后两个反应必需基团的缘故,TPAD~ 虽然作为氢受体有活力,但不能形成Racker band和荧光衍生物。尽管如此,对于绝大多数被测类似物来说,形成荧光衍生物的能力是与它们作为氢受体的活性相平行的,这一事实说明此荧光衍生物的生成,的确发生在活性部位,因此可以用作为研究GAPDH活性部位构象变化的探剂。