Yeast carboxylase

Af-Phenylniacinamide, growth-promoting activity of, VII, 116 Phenylpyruvic acid, effect of yeast carboxylase on, VII, 98 2-Phenylquinoline-4Tcarboxylic acid, diabetogenic action of alloxan and, VII, 371... 

For the determination of thiamine pyrophosphate (the most abundant form of thiamine in animal tissues) a sensitive manometric procedure exists, based upon its function as a coenz une of yeast carboxylase. 

Indeed both -lactylthiamine pyrophosphate (XX) and a-hydroxyethyl-thiamine pyrophosphate (XXI) have been isolated and identified as products after incubation of pyruvate with a purified carboxylase preparation " . When [2- - C]pyruvate is used, the radioactivity is found in the thiazole part of the molecule after sulfite cleavage of XXL Acetaldehyde is formed from pyruvic acid by yeast carboxylase by enzymic cleavage of intermediate XXI, Uberating thiamine pyTophosphate . XXI has also been identified as intermediate in the formation of acetyl-coenzyme A from pyruvic acid by p3u uvic oxidase . The transketolase reaction has been shown to proceed via a gly-colaldehyde-enzyme intermediate here one may expect to find dihydroxy-ethylthiamine pyrophosphate as active glycol-aldehyde . Such experiments strongly support Breslow s concept of the reaction mechanism. 

Carboxylase attacks a large series of a-keto acids, pyruvate most rapidly, and the others at rates that decrease with chain length and other substitutions. The first product of the decarboxylation is the corresponding aldehyde, but in general there is also formation of an acyloin. Yeast carboxylase does not form acetoin from acetaldehyde, but forms... 

In the yeast carboxylase system thiamine and thiamine monophosphate are inactive. Thiamine pyrophosphate reactivates the apoenzyme maximally. It has recently been shown that thiamine triphosphate at a concentration four to five times that of TPP can saturate the apoenzyme giving an activity equivalent to 80% of the TPP reconstituted system. Since thiamine and thiamine monophosphate are inactive, it would appear that a pyrophosphate linkage is necessary for activity. This linkage is assumed to be the site of attachment to the apoenzyme. 

Karrer and Viscontini have found that the thiol form of TPP is as active as TPP in the yeast carboxylase assay. Evidence for the open-ring form of thiamine in nature also is at hand. Approximately 50% of the thiamine in milk has long been known to be present in a form which is released only upon treatment with a proper proteolytic enzyme. In the light of present-day knowledge this bound form is suggestive of a thiamine-protein-S-S complex or of lipothiamide. Bonvicino and Hennessey have prepared a complex of the former type and found it to be biologically active to the extent of 90 %. Myrback and his co-workers have evidence to the effect that well-aerated baker s yeast contains what is probably the disulfide form of TPP. 

Horecker has isolated from spinach leaves a diphosphothiaminopro-tein enzyme which catalyzes the above transketolation between the two pentosephosphates. It is of interest that diphosphothiamine has been shown to be the prosthetic group of all acetoin-forming enzymes such as pyruvic oxidase and yeast carboxylase. 

The results are of comparative interest but may have little bearing on the present discussion because the regulatory mechanisms for microbial fatty acid synthesis and for fatty acid synthesis in animal tissues appear to operate at quite different sites of control. Apart from the obvious absence of primary hormonal signals in bacteria, the following differences stand out. Only animal tissue acetyl-CoA carboxylase is activated by citric acid bacterial, plant and yeast carboxylase do not respond to this type of allosteric modulation. Similarly, microbial acetyl-CoA carboxylases are much more resistant to inhibition by palmitoyl-CoA at least at the concentration which inhibit the hepatic enzyme. 

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