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
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Date
2014-02-06
Authors
ANDREW, Jonathan Nok
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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.
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
Keywords
PURIFICATION,, KINETIC,, STUDIES,, MALATE,, DEHYDROGENASE,, WHITE YAM,, DIOSCOREA ROTUNDATA,, TUBERS