142-10-9Relevant articles and documents
Meyerhof,Kiessling
, p. 43 (1935)
Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population
Fenton, Aron W.,Fenton, Kathryn D.,Lamb, Audrey L.,Martin, Tyler A.,Meneely, Kathleen M.,Swint-Kruse, Liskin,Wu, Tiffany
, (2021/11/20)
Some protein positions play special roles in determining the magnitude of protein function: at such “rheostat” positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A (“I98X”). Despite being ~17 ? from the active site and far from subunit interfaces, substitutions at position 98 have rheostatic contributions to the apparent cooperativity (nH) associated with fructose-1,6-bisphosphate substrate binding and moderately affected binding affinity. Next, we crystallized representative I98X variants to assess structural consequences. Residues smaller than the native isoleucine (cysteine and serine) were readily accommodated, and the larger phenylalanine caused only a slight separation of the two parallel helixes. However, the diffraction quality was reduced for I98F, and further reduced for I98Y. Intriguingly, the resolutions of the I98X structures correlated with their nH values. We propose that substitution effects on both nH and crystal lattice disruption arise from changes in the population of aldolase A conformations in solution. In combination with results computed for rheostat positions in other proteins, the results from this study suggest that rheostat positions accommodate a wide range of side chains and that structural consequences manifest as shifted ensemble populations and/or dynamics changes.
Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase
Zhai, Xiang,Amyes, Tina L.,Wierenga, Rik K.,Loria, J. Patrick,Richard, John P.
, p. 5928 - 5940 (2013/09/23)
Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO 32- complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.
