Enzyme
Architecture: Amino Acid Side-Chains That Function
To Optimize the Basicity of the Active Site Glutamate of Triosephosphate
Isomerase
Version 3 2019-06-04, 08:15
Version 2 2018-06-22, 21:49
Version 1 2018-06-21, 19:43
Posted on 2019-06-04 - 08:15
We report pH rate profiles for kcat and Km for the
isomerization reaction
of glyceraldehyde 3-phosphate catalyzed by wildtype triosephosphate
isomerase (TIM) from three organisms and by ten mutants of TIM; and,
for Ki for inhibition of this reaction
by phosphoglycolate trianion (I3–). The pH profiles for Ki show
that the binding of I3– to TIM (E) to form EH·I3– is accompanied by
uptake of a proton by the carboxylate side-chain of E165, whose function
is to abstract a proton from substrate. The complexes for several
mutants exist mainly as E–·I3– at high pH, in which cases the pH profiles define the pKa for deprotonation of EH·I3–. The linear
free energy correlation, with slope of 0.73 (r2 = 0.96), between kcat/Km for TIM-catalyzed isomerization and the disassociation
constant of PGA trianion for TIM shows that EH·I3– and the
transition state are stabilized by similar interactions with the protein
catalyst. Values of pKa = 10–10.5
were estimated for deprotonation of EH·I3– for wildtype TIM.
This pKa decreases to as low as 6.3 for
the severely crippled Y208F mutant. There is a correlation between
the effect of several mutations on kcat/Km and on pKa for EH·I3–. The results support a model where the strong basicity of
E165 at the complex to the enediolate reaction intermediate is promoted
by side-chains from Y208 and S211, which serve to clamp loop 6 over
the substrate; I170, which assists in the creation of a hydrophobic
environment for E165; and P166, which functions in driving the carboxylate
side-chain of E165 toward enzyme-bound substrate.
CITE THIS COLLECTION
DataCite
3 Biotech
3D Printing in Medicine
3D Research
3D-Printed Materials and Systems
4OR
AAPG Bulletin
AAPS Open
AAPS PharmSciTech
Abhandlungen aus dem Mathematischen Seminar der Universität Hamburg
ABI Technik (German)
Academic Medicine
Academic Pediatrics
Academic Psychiatry
Academic Questions
Academy of Management Discoveries
Academy of Management Journal
Academy of Management Learning and Education
Academy of Management Perspectives
Academy of Management Proceedings
Academy of Management Review
Zhai, Xiang; Reinhardt, Christopher J.; Malabanan, M. Merced; Amyes, Tina L.; Richard, John P. (2018). Enzyme
Architecture: Amino Acid Side-Chains That Function
To Optimize the Basicity of the Active Site Glutamate of Triosephosphate
Isomerase. ACS Publications. Collection. https://doi.org/10.1021/jacs.8b04367
or
Select your citation style and then place your mouse over the citation text to select it.
SHARE
Usage metrics
AUTHORS (5)
XZ
Xiang Zhai
CR
Christopher J. Reinhardt
MM
M. Merced Malabanan
TA
Tina L. Amyes
JR
John P. Richard