A SINGLE AMINO-ACID MUTATION ENHANCES THE THERMAL-STABILITY OF ESCHERICHIA-COLI MALATE-DEHYDROGENASE

The stability of wild-type Escherichia coli malate dehydrogenase was c ompared with a mutant form of the enzyme with the amino acid residue a t position 102 changed from arginine to glutamine. The mutation occurs on the underside of a mobile loop which closes over the active-site c left on formation of the enzyme/cofactor/substrate ternary complex. Th e mutant enzyme is kinetically compromised while the wild-type enzyme is highly specific for oxaloacetate. The mutant enzyme was shown to be more resistant to irreversible thermal denaturation by thermal inacti vation experiments and high-sensitivity differential scanning calorime try than the wild-type enzyme. In contrast, resistance of both enzymes to reversible unfolding in guanidinium chloride was similar. Circular dichroic spectropolarimetry shows the secondary structures of the enz ymes are similar but there is a demonstrable difference in tertiary st ructure. From the position of the mutation, it is conjectured that the substitution on a mobile surface loop results in partial closure of t he loop and greater resistance to thermal inactivation of the mutant e nzyme. However, molecular modelling combined with circular dichroic sp ectropolarimetry indicate that the mutation may have a more widespread effect on the structure than simply partial closure of the mobile sur face loop as the environment of distant tyrosine residues is altered. Resistance of the wild-type enzyme to thermal inactivation can be incr eased by cofactor addition, which may have the effect of partial closu re of the mobile surface loop, but has little effect on the mutant enz yme.