Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein

Abstract

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.

Structural studies of the pigeon cytosolic NADP+‐dependent malic enzyme

“…The N‐terminus of the enzyme, residues 23–31, has large contributions to the dimer interface, and smaller contributions to the tetramer interface. Our previous mutagenesis studies showed that these N‐terminal residues are important for the stability of the enzyme (Chou et al 1997Chou et al 1998; Huang et al 1998). In addition, residue Phe40 is important for the stability of the pigeon enzyme (Chou et al 1996).…”

Electrostatic Contributions to T4 Lysozyme Stability: Solvent-Exposed Charges versus Semi-Buried Salt Bridges

“…Over 20 years ago Perutz (Perutz and Raidt, 1975;Perutz, 1978) noted that salt bridges could potentially increase folding stability. Experimental studies on charge mutations have led to inconsistent conclusions on the contributions of charge-charge interactions to protein stability, and efforts to introduce stabilizing salt bridges have met with mixed success (Anderson et al, 1990;Dao-pin et al, 1991;Sali et al, 1991;Marqusee and Sauer, 1994;Waldburger et al, 1995;Meeker et al, 1996;Tissot et al, 1996;Spek et al, 1998;Vetriani et al, 1998;Huang et al, 1998;Ogasahara et al, 1998;Grimsley et al, 1999;Merz et al, 1999;Ramos et al, 1999;Giletto and Pace, 1999;Loladze et al, 1999;Perl et al, 2000;Pace, 2000;Spector et al, 2000;Strop and Mayo, 2000;Takano et al, 2000;Burkhard et al, 2000;Shaw et al, 2001;Perl and Schmid, 2001;Sanchez-Ruiz and Makhatadze, 2001;Olson et al, 2001;Kammerer et al, 2001;Loladze and Makhatadze, 2002). In contrast, in a number of theoretical studies based on continuum electrostatics, the view appears to have emerged that overall electrostatic interactions destabilize or marginally stabilize proteins and protein complexes (Novotny and Sharp, 1992;Hendsch and Tidor, 1994;Elcock, 1998;Elcock et al, 1999;Sheinerman et al, 2000;…”

Characterization of the functional role of asp 141, asp 194, and asp464 residues in the Mn2+‐l‐malate binding of pigeon liver malic enzyme

“…In the structure-function relationship studies of pigeon liver malic enzyme, we have demonstrated the importance of Lys20 Lys3, Arg9, and Phe19 at the N-terminus of this enzyme for the Mn 2ϩ -l-malate binding and for the subunit association~Chou et al, 1996a, 1996b, 1997, 1998Huang et al, 1998!. The metal binding site of the enzyme was further characterized with metalcatalyzed oxidation~MCO!…”