Enzymes involved in hydroxylations : Inborn errors of metabolism involving Hydroxylation defects & mechanism of aromatic aminoacid Hydroxylations

Abstract

The present studies represent an attempt at inter-institutional collaboration between the Indian Institute of Science, Bangalore and the National Institute of Mental Health and Neurosciences, Bangalore. The investigations reported in this thesis indicate the preliminary studies conducted in this direction and some of the salient findings noted have been summarized.

1. An examination was made of urine samples from 1240 mentally retarded children to detect inborn errors of metabolism. This led to the detection of 14 cases of phenylketonuria. Clinically, they showed mental retardation, delayed milestones of development, brisk knee jerks, and light-colored skin and hair (as compared to the family members). All the cases showed excretion of phenylpyruvic acid and -hydroxyphenylacetic acid in their urine. The serum phenylalanine levels were elevated in all the cases with normal serum tyrosine levels. Parental consanguinity was noted in most of these cases, thus suggesting a recessive mode of inheritance.

2. Apart from the 14 cases of phenylketonuria, 14 other cases of mental retardation associated with other metabolic defects were detected, viz., 3 of homocystinuria, 2 of Hartnup’s disease, and 9 of Hurler’s disease. Cases of homocystinuria showed mental retardation, features of Marfan’s syndrome, and excretion of homocystine in urine. The cases with Hartnup’s disease showed periodic episodes of cerebellar ataxia, photosensitive dermatitis, hyperindoluria, hyperindicanuria, and characteristic generalized hyperaminoaciduria. The cases of Hurler’s disease showed a grotesque facies and other features of gargoylism, and showed increased excretion of dermatan sulfate and heparan sulfate.

3. The total number of cases with metabolic defects detected during this study were 28, which is the first time a large number of such cases is being reported from any one center in India. The other cases reported from India are 8 cases of phenylketonuria, 2 of homocystinuria, 2 of Hartnup’s disease, and 2 of Hurler’s disease.

4. The 14 cases of phenylketonuria (PKU) detected during this study represent more than 50% of such cases reported from India. The other cases of PKU reported are 5 from Vellore, 2 from New Delhi, and 1 from Madras. All the 14 cases detected during this study originate from Mysore State, and one case each from Vellore and Madras also originate from this state.

5. Three cases of PKU have been reported from Pakistan, and of these, one was a case that migrated from Mysore. Thus, out of a total of 25 cases of PKU detected in the Indian subcontinent, 17 originate from Mysore State.

6. One of the causes for the increased prevalence of inborn errors of metabolism is parental consanguinity. However, this does not appear to be the only reason for the high incidence of PKU detected in this series, since consanguinity is widely practiced in South India. A probable reason for the large number of PKU cases detected during this study may be the selection of mentally retarded cases for this study or a careful examination of body fluids to detect inborn errors of metabolism. This does not preclude the consideration of the possibility that a high-risk group for this disease is present in this part of the country.

7. The deficiency of phenylalanine hydroxylase leads to phenylketonuria. In order to understand the mechanism of hydroxylation, investigations on hydroxylation of aromatic amino acids were initiated.

8. A model system consisting of NADH-phenazine methosulfate-O , which generates superoxide anions, was used in this study.

9. Attempts were made to study the hydroxylation using the model system not only of phenylalanine but also of tyrosine and tryptophan. The hydroxylation of tyrosine leads to the formation of DOPA, an important intermediate in cerebral metabolism, and DOPA is also used to alleviate Parkinsonism. Tryptophan is the precursor of serotonin, and impairment of serotonin metabolism has been implicated in the etiology of several neuropsychiatric disorders.

10. Using the model system, it was noted that phenylalanine was converted to tyrosine, tyrosine to DOPA, and tryptophan to 5-hydroxytryptophan. Superoxide anion dismutase, which catalyzes the dismutation of superoxide anions (O + O + 2H H O + O ), was found to inhibit this hydroxylation depending upon the concentration of the dismutase.

11. The inhibition of hydroxylation of the aromatic amino acids by superoxide anion dismutase and the occurrence of this hydroxylation maximally at acid pH suggested the possible involvement of superoxide anions during hydroxylation. Absence of inhibition by xanthine, imidazole, and catalase ruled out the involvement of hydroxyl radicals and hydrogen peroxide in the system.

12. In order to study the involvement of superoxide anions in biological hydroxylations, studies were made on anthranilate hydroxylase isolated from Aspergillus niger. This enzyme converts anthranilate to 2,5-dihydroxybenzoate, and the reaction involves the introduction of two hydroxyl groups into the substrate with elimination of ammonia.

13. Anthranilate hydroxylase was induced by growing Aspergillus niger in a medium containing anthranilate. The enzyme was recovered from the mycelial filtrate into 0.025 M phosphate buffer (pH 7.2) and subjected to protamine sulfate treatment, negative adsorption on DEAE-cellulose, ammonium sulfate fractionation, and positive DEAE treatment.

14. The enzyme, which gave a single band on polyacrylamide gel electrophoresis, had a pH optimum of 8.2 and a temperature optimum of 32°C. The enzyme required both anthranilate and NADPH for activity and did not utilize 3-hydroxyanthranilate as substrate. The Km for anthranilate was 0.12 mM, and for NADPH it was 0.14 mM. The Vmax for the enzyme was 0.14 µmole of 2,5-dihydroxybenzoate formed per mg protein per minute.

15. The enzyme required NADPH for activity. It was noted that ammonia, one of the products during enzyme activity, acted as a competitive inhibitor of anthranilate. NADP with respect to both NADPH and anthranilate was found to be non-competitive. The reaction appeared to proceed in an ordered mechanism.

16. Among the various metal chelators tested, o-phenanthroline showed maximum inhibition of enzyme activity, and with varying concentrations of the inhibitor it was noted that it acted as a non-competitive inhibitor of the enzyme.

17. Several sulfhydryl inhibitors, including sodium arsenite which interacts with dithiols, inhibited the enzyme activity. PCMB (p-chloromercuribenzoate) was found to be a very potent inhibitor of the enzyme activity. The inhibition was found to be reversible by mercaptoethanol.

18. It was noted that oxidation of NADPH by the enzyme was dependent on the presence of anthranilate, since no oxidation of NADPH occurred in the absence of anthranilate.

19. The NADPH oxidation could be linked to several electron acceptors such as cytochrome-c and NBT. It was noted that in the absence of anthranilate there was no reduction of NBT by the enzyme. With increasing concentrations of anthranilate, a decreased reduction of NBT was noted, which suggested that possibly both the dye and the substrate were competing for the same substrate, viz., superoxide anion. The enzyme was inhibited by superoxide anion dismutase.

An interesting observation during purification of the enzyme was a large increase in total enzyme activity during early stages of purification, suggesting removal of an inhibitor. The DEAE precipitate, at this stage of purification where an increase of recovery was noted, was extracted into 0.25 M phosphate buffer and when this was added to the model system it inhibited the reduction of NBT. These preliminary results suggest that the inhibitor may be superoxide anion dismutase.

20. Investigations presented in this thesis show the possible presence of a high-risk group for phenylketonuria in Mysore State and also indicate the involvement of superoxide anions in the hydroxylation of aromatic amino acids.