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Engineering of a stable mutant malic enzyme by introducing an extra ion‐pair to the protein
Author(s) -
Huang ShihMing,
Chou WeiYuan,
Lin ShuIu,
Chang GuGang
Publication year - 1998
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19980401)31:1<61::aid-prot6>3.0.co;2-k
Subject(s) - denaturation (fissile materials) , chemistry , urea , enzyme , enzyme assay , enzyme kinetics , methionine , mutant , malic enzyme , arginine , kinetics , reaction rate constant , crystallography , biochemistry , amino acid , analytical chemistry (journal) , chromatography , nuclear chemistry , active site , quantum mechanics , gene , dehydrogenase , physics
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°C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58°C also conformed to a three‐state model with the rate constant for the intermediate state of R9E/M17K ( k 2 = 0.03 min ‐1 ) being much smaller than the WT value ( k 2 = 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 m Mn and K m Mal by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [ k cat /( K m Mn K m NADP K m Mal )] 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. Proteins 31:61–73, 1998. © 1998 Wiley‐Liss, Inc.