The binding of substrates to lactate dehydrogenases induces a marked rearrangement of the protein structure in which a ‘loop’ of polypeptide (residues 98–110) closes over the active site of the enzyme1,2. In this rearrangement, arginine 109 (a basic residue conserved in all known lactate dehydrogenase sequences and in the homologous malate dehydrogenases3) moves 0.8 nm from a position in the solvent4 to one in the active site1,5,6 where its guanidinium group resides within hydrogen bonding distance of both the reactive carbonyl of pyruvate and imidazole ring of the catalytic histidine 195 (see Fig. 1). Whilst this feature of the enzyme has been commented upon previously1, the function of this mobile arginine residue during catalysis has not been tested experimentally. The advent of protein engineering has now enabled us to define the role of this basic residue by substituting it with the neutral glutamine. Transient kinetic and equilibrium studies of the mutant enzyme indicate that arginine 109 enhances the polarization of the pyruvate carbonyl group in the ground state and stabilizes the transition state. The gross active-site structure of the enzyme is not altered by the mutation since an alternative catalytic function of the enzyme (rate of addition of sulphite to NAD+), which does not require hydride transfer, is insensitive to the arginine→glutamine substitution.
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Clarke, A., Wigley, D., Chia, W. et al. Site-directed mutagenesis reveals role of mobile arginine residue in lactate dehydrogenase catalysis. Nature 324, 699–702 (1986). https://doi.org/10.1038/324699a0
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