Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.456868
Title: Studies on soluble and immobilized NADP⁺ specific enzymes
Author: Goheer, Mohammad Anwar
ISNI:       0000 0001 3501 6836
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1974
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Abstract:
Baker's yeast glucose 6-phosphate dehydrogenase (G6PD) (D-glucose 6-phosphate: nicotinamide-adenine dinucleotide phosphate oxido-reductase, EC 1. 1. 1. 49) was immobilized on DE 52-cellulose by the triazine method and on Sephadex, Sepharose and Sepharose derivatives by the cyanogen bromide and carbodiimide methods. The properties of these preparations were studied using packed-bed and gradientless recirculation reactors. Sepharose-G6PD, valine Sepharose-G6PD and ethylene diamine Sepharose-G6PD preparations were stable on prolonged storage. They were completely stable over the pH range studied (6-10) and were also more heat stable than the soluble G6PD. The optimum pH of valine Sepharose-G6PD and ethylene diamine Sepharose-G6PD were slightly shifted towards alkaline and acidic regions respectively when compared to soluble and Sepharose-G6PD. The effect of pH on the reaction equilibrium of soluble and immobilized enzymes was also investigated. The kinetics of this enzyme had not been investigated previously, so detailed studies on soluble and immobilized enzymes were carried out. The Michaelis constants of Sepharose-G6PD (K[m][G6F] = 1. 2 x 10[-4]M and K[m][NADP+] = 4.5x10[-5]M) were similar to those of the soluble enzyme (K[m][G6P] = 0.5x10[-4]M and K[m][NADP+] = 1.9x10[-5]M). Soluble (K[s][NADPH] = 1.2 x 10[-5] M) and immobilized (K[s][NADPH] = 2.0x10[-5]M) enzymes were competitively inhibited by NADPH when NADP[+] concentration was varied. Non-competitive inhibitions were shown by both soluble (K[is] = 7.6 x 10[-5]M) and immobilized (K[is] = 3.1x10[-5]M) enzymes when the concentration of G6P was varied in the presence of NADPH. ATP was a non-competitive inhibitor with the soluble (K[is] = 2.2x10[-3]M) as well as with the immobilized (K[is]= 4.0 x 10[-3]M) enzyme in the presence of a constant concentration of G6P but in the presence of constant NADP[+] concentrations, ATP gave S-parabolic competitive inhibition. Both soluble and immobilized enzymes apparently gave an ordered Bi Bi reaction pathway in which NADP[+] and NADPH were the leading reactants. NAD[+] kinase (ATP:NAD 2'-phosphotransferase, EC 2.7.1.23) was purified from Azotobacter chroococcum but a stable immobilized enzyme was not prepared.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.456868  DOI: Not available
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