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 denaturation, but was enzymatically less active than the wild-type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased 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 intermediate state of R9E/M17K (k2 = 0.03 min(-1)) being much smaller than the WT value (k2 = 2.39 min(-1)). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the K(mMn) and K(mMal) by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [k(cat)/(K(mMm)K(mNADP)K(mMal))] for the R9 mutants at least four orders of magnitude smaller than the WT. M17K has similar properties to the WT, while R9E is more labile than the WT enzyme. The above results indicate that the extra stability gained 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 acid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue. Since the R9E mutant, which has an extra Glu9-Lys15 ion-pair, is less stable than the WT, we conclude that the contribution to malic enzyme stability is specific for the Glu9-Lys17 ion-pair.