Role of Lys-12 in catalysis by triosephosphate isomerase: a two-part substrate approach.
We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a approximately 50-fold increase in K(m) for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K(i) for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k(cat) for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 x 10(5)-fold decrease in k(cat)/K(m), corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D(2)O were quantified as dihydroxyacetone phosphate (DHAP) (27%), [1(R)-(2)H]DHAP (23%), [2(R)-(2)H]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D(2)O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >or=14-fold decrease in k(cat)/K(m) for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-(13)C]GA in D(2)O is [1-(13)C,2,2-di-(2)H]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k(cat)/K(m) for the reactions of both GAP and the neutral truncated substrate [1-(13)C]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".
Go
Maybelle K
MK
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260-3000, USA.
Koudelka
Astrid
A
Amyes
Tina L
TL
Richard
John P
JP
D001483:Base Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D014305:Triose-Phosphate Isomerase; D008239:Lysine