The objective of this thesis was to determine the molecular basis of neonatal multiple carboxylase deficiency (MCD) produced by an impairment in holocarboxylase synthetase (HCS) activity and the origin of the biotin-responsiveness that characterizes this disease. To determine HCS activity, I developed a peptide substrate and used the biotinylation system of E: coli to determine its properties. C-terminal fragments of the $ alpha$ subunit of human propionyl-CoA carboxylase (PCC-$ alpha$) were expressed in E. coli and site-directed mutagenesis was used to define the residues required for biotinylation by the bacterial biotin ligase, BirA. These experiments showed that the biotin region of PCC-$ alpha$ can act as an autonomous domain for biotinylation and suggested its use as substrate for human HCS. For the molecular characterization of MCD, I isolated several cDNA clones encoding human HCS by functional complementation of an E. coli mutant with a temperature-sensitive BirA. Comparison of the predicted amino acid sequence of HCS with bacterial biotin ligases allowed the identification of the putative biotin-binding domain of this protein. Mutation analysis of DNA from HCS deficient patients showed that most of the changes in the HCS sequence are clustered in the biotin-binding domain. All the patients tested in this study showed deficiency of HCS activity as determined using the PCC-$ alpha$ peptide as substrate for biotinylation. The biotin-responsiveness was demonstrated by obtaining a stimulation of HCS activity of MCD cells at high biotin concentrations while remaining unstimulated in extracts of normal cells. Together with the mutation studies, these results showed that neonatal MCD is caused by mutations in the biotin binding domain of HCS which reduce the affinity of the enzyme towards biotin. This change in the kinetic properties of HCS results in the inefficient biotinylation of carboxylases at physiological concentrations of biotin. The defect can be over

Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of three mitochondrial and one cytosolic forms of biotin-dependent carboxylases in humans. Patients suffering from this autosomal recessive disease have Multiple Carboxylase Deficiency (MCD) with symptoms of life-threatening metabolic acidosis which, in almost all cases, can be successfully treated with pharmacologic doses of oral biotin. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS maybe targeted to the cytoplasm and mitochondria or that carboxylases are biotinylated in the cytoplasm prior to import into the mitochondria. In order to resolve the compartmentalization of HCS, 5' HCS cDNA sequences have been examined for a targeting signal and a candidate sequence was tested for its capacity to target mitochondria. Analysis of 5' cDNA reveals complex alternative splicing, none of which appear to contain mitochondrial targeting sequences. In addition, antibodies have been developed in order to perform immunochemical analysis of the subcellular distribution of HCS. Polyclonal antisera were raised against full length HCS as well as two peptides corresponding to a 20 amino acid region in the N-terminus and to the 20 amino acids preceding the stop codon. Immunohistochemical staining of human fibroblasts with the antibody to full length HCS gives cytosolic, mitochondrial and nuclear localization. Interestingly, analysis with the N-terminal antiserum reveals a large punctate staining pattern exclusively localized to the nucleus. The corresponding C-terminal antiserum reveals solid nuclear staining with some mitochondrial co-localization. Taken together, these results indicate the ubiquitous nature of HCS in human cells and also allude to a potential role for HCS in the nucleus of human cells.

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Thesis (Ph. D.)--University of Michigan, 1930. / Caption title: Comparative studies of the metabolism of the amino acids. V. The oxidation of phenylalanine and phenylpyruvic acid in the organism of the rabbit, by Joseph P. Chandler and Howard B. Lewis. "Reprinted from the journal of biological chemistry, vol. XCVI, no. 3 ... June, 1932." Bibliography: p. 635-636.

Thesis (Ph. D.)--University of Michigan, 1930. / Caption title: Comparative studies of the metabolism of the amino acids. V. The oxidation of phenylalanine and phenylpyruvic acid in the organism of the rabbit, by Joseph P. Chandler and Howard B. Lewis. "Reprinted from the journal of biological chemistry, vol. XCVI, no. 3 ... June, 1932." Bibliography: p. 635-636.

My thesis is divided into 4 major sections. The first section is devoted in part to a description of the biochemical abnormalities in the metabolism of homocarnosine (y-aminobutyryl-L-histidine, HCarn) occurring in a patient with homocarnosinosis. The patient studied and two of her siblings have a progressive neurological disorder with grossly elevated concentrations of HCarn in their CSF. HCarn content was four times higher in a biopsy from the patient's frontal cortex than in biopsied cortex from a large group of control subjects. Using new techniques for the measurement of the HCarn synthesizing and catabolizing enzymes, it was found that the activity of HCarn-Carn synthetase was not increased in the patient's biopsy whereas homocarnosinase activity was undetectable. It is concluded that the elevated HCarn in brain and CSF in the homocarnosinosis patient is due to a deficiency of brain homocarnosinase activity. The first description of the regional distribution of the two HCarn metabolizing enzymes in human brain was also obtained. The remainder of the first section deals with a description of the neuropharmacological properties of HCarn. Intraventricular injection of HCarn in the rat produced hyperexcitability and in high doses, convulsions, whereas unilateral intra-striatal injection of HCarn resulted in contralateral myoclonus. The results of these experiments are consistent with the possibility that HCarn may be involved in the neuronal excitability of brain. The second section describes experiments which test the hypothesis that the content of the inhibitory neurotransmitter GABA is altered in the autopsied brains of some patients dying with schizophrenia. The mean content of GABA was reduced by 20-25% in nucleus accumbens, caudate nucleus, frontal cortex and thalamus of the schizophrenic patients as compared to a control group. However, the differences were found to be statistically significant for only the caudate and thalamus. Extraneous factors such as age of patient at death and prolonged drug treatment did not readily explain the observed reduction in GABA content. The results of the investigation suggest an association between a deficiency of GABAergic function in certain brain areas with some forms of schizophrenia. The third section describes experiments which test the hypothesis that a deficiency of aspartate found in the cerebellar cortex of some patients with dominantly inherited cerebellar disorders might be due to reduced activity of two enzymes involved in the synthesis of aspartate, namely, aspartate aminotransferase and pyruvate carboxylase. No deficiency of either enzyme was observed in the cerebellar specimens studied. The results of this investigation suggest that the aspartate deficiency in cerebellar cortex found in some dominantly inherited cerebellar disorders does not result from a deficiency of either of these two brain enzymes. In the final section, experiments are described which study the effects of chronic administration of Y-vinyl GABA and of hydrazine on the contents of GABA and other amino compounds in rat brain. Both of these compounds are presently under consideration for use in clinical trials on patients with disorders involving a brain GABA deficiency. Chronic administration of either y-vinyl GABA or of hydrazine markedly increased brain GABA content in the rat. Prolonged treatment with y-vinyl GABA, but not hydrazine, produced a decrease in the activity of glutamic acid decarboxylase (GAD) in rat brain. Since GAD is localized to a large extent in nerve endings, the possibility exists that y-vinyl GABA might reduce the amount of GABA available for release at synapses, a potentially undesirable effect. The contents of many brain amino compounds other than GABA were markedly altered by both drugs. Since the potential harmful effects of these unexpected biochemical alterations in brain are unknown, the nonspecific effects of Y-vinyl GABA and hydrazine are disturbing. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

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