Subject Area

Chemistry

Description

Cancer cells preferentially undergo glycolysis in aerobic environments, a phenomenon termed the Warburg effect. Malate dehydrogenase (MDH) catalyzes the reversible interconversion of malate and oxaloacetate. Human cytosolic malate dehydrogenase (hMDH1) isoform 3 is involved in the malate-aspartate shuttle (MAS), which oxidizes cytosolic NADH. hMDH1 is implicated in high aerobic glycolysis in cancer cells because NAD is a necessary cofactor for glycolysis. Thus, hMDH1 is a promising molecular target for cancer treatment. A single proline residue at position 110 in the mobile active site loop of hMDH1 was mutated to a serine with the intention of altering the enzyme’s substrate specificity. Steady-state kinetics analysis showed that mutant activity with the native substrate, oxaloacetate, exceeded that of the wildtype (WT) enzyme (kcat of 320 ± 10 s-1 and 780 ± 30 s-1 for the WT and mutant, respectively). Catalytic activity of both enzymes decreased significantly with the alternative substrate, α-ketoglutarate (kcat of 3 ± {6} s-1 and 0.19 ± 0.01 s-1 for the WT and mutant, respectively). The specificity constant (ksp = kcat /KM) signifies the efficiency of an enzyme for competing substrates. The ksp values for the WT and mutant with OAA were 1.7 ± 0.2 × 107 M-1s-1 and 8.21 ± 0.46 ×106 M-1s-1, respectively. These ksp values were substantially higher than those for both enzymes with α-ketoglutarate. We can conclude that the WT and mutant hMDH1 enzymes function optimally with oxaloacetate. Also, Pro110Ser hMDH1 has altered substrate specificity, with α-ketoglutarate functioning as an alternative substrate.

Publisher

Providence College

Date

Spring 2022

Type

Article

Format

Text

.pdf (text searchable)

Language

English

Included in

Biochemistry Commons

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