PURIFICATION AND SOME KINETIC STUDIES OF MALATE DEHYDROGENASE FROM WHITE YAM (DIOSCOREA ROTUNDATA) TUBERS
PURIFICATION AND SOME KINETIC STUDIES OF MALATE DEHYDROGENASE FROM WHITE YAM (DIOSCOREA ROTUNDATA) TUBERS
| dc.contributor.author | ANDREW, Jonathan Nok | |
| dc.date.accessioned | 2014-02-06T09:57:20Z | |
| dc.date.available | 2014-02-06T09:57:20Z | |
| dc.date.issued | 2014-02-06 | |
| dc.description | A THESIS SUBMITTED TO THE POSTGRADUATE SCHOOL, AHMADU BELLO UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCE AHMADU BELLO UNIVERSITY ZARIA - NIGERIA | en_US |
| dc.description.abstract | Malate dehydrogenases [L-Malate MAD+ oxidoreductase E.C. 1.1.1.371 have been purified from white yam (Diosoorea rotundata) tuber by a p u r i f i c a t i o n protocol involving ammonium sulphate fractionation, ion exchange chromatography on DEAE-cellulose, and Sephadex gel chromatography. The enzyme was resolved into two forms MDH1 and MDH2 on DEAE-cellulose. Both enzyme forms were homogenous with different electrophoretic mobilities on polycrylamide gels. Their mobilit i e s on S.D.S.-polycrylamide gel were however the same. The molecular weights of the yam tuber malate dehydrogenases determined by gel f i l t r a t i o n were 61,300 ± 150 and 63,000 ± 400 for MDH1 and MDH2 respectively while the subunit molecular weight of 31,000 was determined by SDS-PAGE for both enzymes. The a c t i v a t i o n energy (Ea) and Arhenius constant (A) for MDH1 were 33.bkJ/mole and 1.94 x 105dm3m mole-1min in the same respect, the values for MDH2 were 47.8kJ/mole and 4.92.x 107dm3m mole-1min". The MDH1 showed two pH maxima at 6.5 and 7.5 assayed at 30°C. Groups associated with enzyme c a t a l y s i s and binding of substrate have pKa values of 6.8 and 7.6. A single pH maximum of 6.5 associated to pKa of 6.8 was manifested by MDH2. Both forms of the enzyme were inhibited by oxaloacetic acid concentration at or greater than 0.25mM at pH 7.5. The half l i fe of MDH.1 was found to be 122 days in the freezer, 20 days in the refrigerator, and 8.3 days at ambient temperature. The half l i fe of MDH2 in the freezer, refrigerator and at ambient temperature were: 5 days, 31/2 days and 11 /4 days respectively. The i n i t i a l velocity studies with the enzymes indicated an ordered sequential mechanism. The Km values obtained from secondary plots were 0.05mM 0.08mM 0.48mM, 2.56mM for NADH, OAA, NAD+ and 1-malate respectively for MDH1. The values fcr HDH2 were found to be 0.02mM, 0.083mM, 0.25mM and 0.83mM in the same respect. Product i n h i b i t i o n analysis in both forward and backward reactions to decipher the kinetic meohanism for MDH1 revealed an ordered bi-bi sequential-mechanism. ATP i n h i b i t i o n on both enzymes was competitive with respect to OAA. Both enzymes were inhibited by naturally occuring metabolites like -ketoglutarate, c i t r a t e , suooinate. Inhibition studies with group specific reagents: Parahydroxy meroury benzoate, KCN, NaN,, strongly inhibited both enzymes, thus implicating the thiol group (-SH) in the catalytic potency of the enzyme. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/397 | |
| dc.language.iso | en | en_US |
| dc.subject | PURIFICATION, | en_US |
| dc.subject | KINETIC, | en_US |
| dc.subject | STUDIES, | en_US |
| dc.subject | MALATE, | en_US |
| dc.subject | DEHYDROGENASE, | en_US |
| dc.subject | WHITE YAM, | en_US |
| dc.subject | DIOSCOREA ROTUNDATA, | en_US |
| dc.subject | TUBERS | en_US |
| dc.title | PURIFICATION AND SOME KINETIC STUDIES OF MALATE DEHYDROGENASE FROM WHITE YAM (DIOSCOREA ROTUNDATA) TUBERS | en_US |
| dc.type | Thesis | en_US |