514 2. ANALOGS OF ENZYME KEACTION COMPONENTS 



kaline pH to generate a free SH group. The NAD analog with 4-methyl-5- 

 (/?- hydroxy ethyl )thiazole replacing nicotinamide was found to behave in 

 this manner and to form a disulfide bond with SH groups at the site of 

 dehydrogenases, this binding being competitive with NAD. In the case of 

 horse liver alcohol dehydrogenase, 2 moles of this analog are bound tightly 

 to each mole of enzyme. 



One of the most interesting studies of dehydrogenase inhibition by nu- 

 cleotides is that of the complex effects of GTP on glutamate dehydrogenase 

 (Frieden, 1962, 1963). The K^ is 0.0003-0.0005 mM and the kinetics being 

 uncompetitive point to different sites for NADP and GTP. Furthermore, 

 GTP not only inhibits directly but increases the ability of NADH to inhibit. 

 Since the NADH inhibition is due to the dissociation of the enzyme into 

 four subunits, it is likely that GTP enhances the process, and this was dem- 

 onstrated ultracentrifugaUy. The dissociation of the tetramer enzyme it- 

 self is not necessarily the basic cause of the loss of activity; it is possible 

 that structural changes brought about by NADH and GTP produce both 

 dissociation and reduced catalytic activity. The behavior can be explained 

 adequately on the basis of three binding sites: (1) a coenzyme site, (2) a 

 purine nucleotide site with which GTP and activating nucleotides react, 

 and (3) a NADH-binding site. The following complexes are thus possible 

 — EC, ECI, ECg, ECgl, and EI — where C represents the coenzyme. The 

 binding of GTP to the enzyme depends on the presence of NADP at a 

 vicinal site, the EI complex probably not being of much importance. The 

 importance of this situation for the regulation of cell metabolism is obvious, 

 particularly since this enzyme plays a central role in many pathways. Frie- 

 den pointed out the likely relationship between glutamate dehydrogenase 

 and the or-ketoglutarate step in the cycle; GDP is required for the conver- 

 sion of succinyl-CoA to succinate and GTP is formed, which can suppress 

 the activity of glutamate dehydrogenase, an enzyme which under certain 

 conditions controls the steady-state level of a-ketoglutarate in the cycle. 

 He also suggests that ammonia formation by the liver, protein synthesis, 

 and glyconeogenesis can all be regulated by this inhibition involving a feed- 

 back site. 



ANALOGS OF THIAMINE 



Thiamine functions in metabolism in the pyrophosphorylated form as the 

 coenzyme in various reactions where a bond adjacent to a carbonyl group is 

 broken (a-cleavage), the active complex in each case being an aldehyde- 

 thiamine-PP-enzyme structure wherein a C — C bond is formed at the 2- 

 position of the thiazole ring. These reactions would include (1) a-keto acid 

 decarboxylation (e.g. pyruvate decarboxylase), (2) a-keto acid oxidation 

 (e.g. pyruvate and a-ketoglutarate oxidases), (3) the phosphoroclastic reac- 

 tion of pyruvate, and (4) a-ketol formation (e.g. transketolase and phos- 



