Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)




The branched-chain amino acids leucine, isoleucine and valine are transaminated to their respective a-keto acid analogues; a-keto- isocaproate (KIC) a-keto-p-methyIvalerate (KMV) and a-ketoiso- valerate (KIV) in normal mammalian tissues. Recently an interest in the genetic anomaly "branched-chain ketoaciduria" or "maple syrup urine disease", a metabolic disease involving the oxidative decarboxylation of the three branched-chain CC-keto acids, has stimulated efforts to characterize the nature of the enzymic conversions leading from the a-keto acids to their acyl coenzyme A products. These studies have been extended toward a consideration of the metabolic interrelationships among these a-keto acids (KIC, KMV, KIV). Furthermore, the effects of the branched-chain a-keto acids upon the metabolism of pyruvate and of a-ketoglutarate have been investigated. In addition, there has been an attempt to determine whether, in the normal and the abnormal (branched-chain ketoaciduric) animal, such interrelationships might constitute a significant physiological regulatory mechanism.

a-Keto acid dehydrogenase activities were estimated by the Ik quantitative measurement of CO^ evolved from carboxyl-labeled a- keto acid substrates. The a-keto acid dehydrogenase enzyme activities were measured in several fractions of bovine liver homogenates prepared by differential centrifugation techniques.

Subcellular distribution studies established that the branched- :hain a-keto acid dehydrogenase activities were localized principally „n the mitochondri ., and more specifically on the outside of the inner membrane of the mitochondria. In addition to the particulate activity, minor amounts of dehydrogenase activity with KMV and KIC /ere noted in the "soluble" (not sedimented at 80,000 x £ for one lour) fraction.

The particulate branched-chain a-keto acid dehydrogenase activities were shown to depend upon added nicotinamide adenine linucleotide (NAD+) and coenzyme A for maximal activity. Also jbserved were apparent requirements fox calcium, magnesium and Inorganic phosphate. These findings lend support to the assumption :hat the branched-chain a-keto acids are catabolized by a mechanism analogous to that known to exist for the well-characterized ayruvate and a-ketoglutarate dehydrogenase complexes.

The apparent Michaelis constant for each of these keto acids KIC, KMV, KIV, pyruvate, a-ketoglutarate) was determined. In general, the values for the branched-chain a-keto acids are of the . -h >rder of 4 x 10 M, while those for pyruvate and a-ketoglutarate -ij. -I4. ire slightly lower, 0.8 x 10 M and 2 x 10 M respectively.

The degree and type of influence exerted among the various a- :eto acids was examined directly by following the enzymatic lip .iberation of CO^ from carboxyl-labeled substrate in the presence if an alternate unlabeled a-keto acid. A mutually inhibitory iattern among KIC, KIV and KMV was noted. In addition, each of the •ranched-chain keto acids exerted a marked inhibition of pyruvate dehydrogenase and a-ketoglutarate dehydrogenase activities. Inhibitions were of the competitive type in all instances except for a-ketoglutarate dehydrogenase activity, where a "mixed" type of inhibition was seen. The apparent inhibitor constants were in a range such that a significant mutual influence among the branched- chain a-keto acids could exist in vi ^o. Also, the inhibitory effects of KIC upon pyruvate dehydro '.anase could be considered to be significant at normal cellular concentrations of these metabolite The net effect of the inhibitory action of the branched-chain a-keto acids on each other and upon pyruvate dehydrogenase in the normal animal is likely a homestatic balance, which allows for catabolism of excess branched-chain amino acids, while concurrently influencing the flow of pyruvate to acetyl coenzyme A.

The abnormal situation, as obtained in branched-chain keto aciduria, results in extremely elevated levels of all three branched chain a-keto acids. As these acids accumulate proximal to the metabolic block at the level of oxidative decarboxylation, they may nearly completely restrict the activity of pyruvate dehydrogenase, and exert substantial inhibitory effects at a-ketoglutarate dehydrogenase. These effects could severely limit the production of energy via the Krebs tricarboxylic acid cycle. In addition, production of acyl coenzyme A intermediates necessary for fatty acid synthesis would be affected.

Thus, as the result of a genetic.lesion, which is manifest in the inability of the animal to catabolize the branched-chain a-keto acids, these intermediates accumulate, and in turn may exert a deleterius influence upon cellular energy requirements, as well as on production of essential metabolic intermediates. These effects in the abnormal situation could account for many of the symptoms observed in branched-chain ketoaciduria.