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Defining the fumarase water binding site
Author(s) -
Weaver Todd Michael
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.796.18
Subject(s) - fumarase , active site , chemistry , binding site , aspartic acid , glutamine , stereochemistry , alanine , allosteric regulation , crystallography , amino acid , enzyme , biochemistry
Fumarase C, a class II family member, catalyzes the nearly catalytically perfect reversible conversion of fumarate to S‐malate as part of the Krebs Cycle. Despite decades of research and high quality structural information the exact mechanism for diffusion limited catalysis remains elusive. In order to more fully understand the contribution of various active site residues to the fumarase‐catalyzed reaction, a series of crystal structures and kinetic parameters for a series of active site fumarase variants have been determined . The first set of variants targeted the water 26 (W26) binding site formed via Ser98, Thr100, Asn141 and His188. Alterations at the water‐binding site, termed the W26 cluster, affected the K m and k cat . The structural data for each W26 cluster replacement notes only slight shifts in this structural water location with maximal movement of 4.9 Å in the H188A active site leading to an overall 3500‐fold reduction in activity. The second set of variants replaced the highly conserved glutamic acid at position 331 with either an aspartic acid or a glutamine residue. Glu331, contained within the fumarase signature sequence, forms a shortened hydrogen bond to stabilize His188 just behind the W26 cluster. The Glu331 to aspartic acid or glutamine substitution create significant reductions in k cat and limited effect on K m values even though there are few structural changes in the position of W26. Based upon the structural and kinetic data we propose the following (1) W26 provides a model for both the S‐malate 2‐[OH] binding site during the S‐malate dehydration reaction and the water binding site during the fumarate hydration reaction, (2) Asn141 and Glu331 facilitate Nδ1‐H to Nɛ2‐H tautomeric interconversion of His188 and (3) His188 acts as the catalytic acid to protonate the S‐malate 2‐[OH] during the S‐malate à fumarate dehydration. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .