ENGINEERING OF A STABLE MUTANT MALIC ENZYME BY INTRODUCING AN EXTRA ION-PAIR TO THE PROTEIN

A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17 , respectively, was found to be much more stable in urea and thermal d enaturation, but was enzymatically less active than the wild-type enzy me (WT), Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was com pletely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermedia te was involved in the denaturation process, The half unfolding urea c oncentration, measured by fluorescence spectral changes, increased fro m 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increa sed by approximately 10 degrees C for R9E/M17K compared with that for WT, Kinetic analysis of the thermal inactivation at 58 degrees C also conformed to a three-state model with the rate constant for the interm ediate state of R9E/M17K (k(2) = 0.03 min-l) being much smaller than t he WT value (k(2) = 2.39 min(-1)). Results obtained from single mutant s indicated that the decreasing enzyme activity of R9E/M17K was exclus ively due to R9 mutation, which increased the K-mMn and K-mMal by at l east one order of magnitude compared with WT, Consequently, a decrease occurred in the specificity constant [k(cat)/(KmMnKmNADPKmMal)] for t he R9 mutants at least four orders of magnitude smaller than the WT, M 17K has similar properties to the WT, while R9E is more labile than th e WT enzyme, The above results indicate that the extra stability gaine d by the double mutant possibly occurs through the introduction of an extra ion-pair between E9 and K17, which freezes the double mutant in the putative intermediate state. Examination of the N-terminal amino a cid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue, Since the R9E mutant, which has an extra Glu9-L ys15 ion-pair, is less stable than the WT, we conclude that the contri bution to malic enzyme stability is specific for the Glu9-Lys17 ion-pa ir. (C) 1998 Wiley-Liss, Inc.