Rescue of K12G triosephosphate isomerase by ammonium cations: the reaction of an enzyme in pieces.
The K12G mutation at yeast triosephosphate isomerase (TIM) results in a 5.5 × 10(5)-fold decrease in k(cat)/K(m) for isomerization of glyceraldehyde 3-phosphate, and the activity of this mutant can be successfully "rescued" by NH(4)(+) and primary alkylammonium cations. The transition state for the K12G mutant TIM-catalyzed reaction is stabilized by 1.5 kcal/mol by interaction with NH(4)(+). The larger 3.9 kcal/mol stabilization by CH(3)CH(2)CH(2)CH(2)NH(3)(+) is due to hydrophobic interactions between the mutant enzyme and the butyl side chain of the cation activator. There is no significant transfer of a proton from alkylammonium cations to GAP at the transition state for the K12G mutant TIM-catalyzed reaction, because activation by a series of RNH(3)(+) shows little or no dependence on the pK(a) of RNH(3)(+). A comparison of k(cat)/K(m) = 6.6 × 10(6) M(-1) s(-1) for the wildtype TIM-catalyzed isomerization of GAP and the third-order rate constant of 150 M(-2) s(-1) for activation by NH(4)(+) of the K12G mutant TIM-catalyzed isomerization shows that stabilization of the bound transition state by the effectively intramolecular interaction of the cationic side chain of Lys-12 at wildtype TIM is 6.3 kcal/mol greater than that for the corresponding intermolecular interaction of NH(4)(+) at K12G mutant TIM.
Go
Maybelle K
MK
Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, USA.
Amyes
Tina L
TL
Richard
John P
JP
D002384:Catalysis; D002412:Cations; D008958:Models, Molecular; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*
D002412:Cations; D000644:Quaternary Ammonium Compounds; D014305:Triose-Phosphate Isomerase
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