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The Complex Role of Asn270 in Dianion Activation of Glycerol 3‐Phosphate Dehydrogenase‐Catalyzed Hydride Transfer
Author(s) -
Reyes Archie C.,
Amyes Tina L.,
Richard John P.
Publication year - 2016
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.30.1_supplement.lb114
Subject(s) - dihydroxyacetone phosphate , chemistry , dhap , dehydrogenase , triosephosphate isomerase , hydride , stereochemistry , glycerol 3 phosphate dehydrogenase , catalysis , active site , substrate (aquarium) , enzyme , substrate analog , medicinal chemistry , photochemistry , biochemistry , organic chemistry , hydrogen , oceanography , geology
We are working to understand the mechanism for the strong phosphite dianion (HP i ) activation of proton transfer reactions catalyzed by triosephosphate isomerase (TIM), decarboxylation reactions catalyzed by orotidine monophosphate decarboxylase (OMPDC), and hydride transfer reactions catalyzed by glycerol 3‐phosphate dehydrogenase (GPDH). The phosphodianion of substrate dihydroxyacetone phosphate (DHAP) for GPDH provides 11 kcal/mol stabilization of the transition state for hydride transfer. Eight kcal/mol of this stabilization is observed as HP i activation of GPDH for catalysis of the reduction of glycolaldehyde (GA). The X‐ray crystal structure of the nonproductive complex between GPDH, DHAP and NAD + shows interactions of Arg269 and Asn270 with the substrate phosphodianion. The R269A mutation of GPDH results in a large 9.1 kcal/mol destabilization of the transition state for enzyme‐catalyzed reduction of DHAP from elimination of the enzyme‐substrate ion‐pair; and 6.7 kcal/mol of this stabilization is recovered when the reaction is carried out in the presence of 1.0 M guanidinium cation.1 Similar effects were reported for K12G and R235A mutations of TIM2 and OMPDC,3 respectively, consistent with a similar architecture of the dianion activation sites for these enzymes. However, the following effects of N270A and R269A/N270A mutations require an unexpected greater mechanistic complexity for dianion activation of GPDH, compared with OMPDC and TIM. 1.) The 5.6 kcal/mol effect from N270A mutation on transition state stability is more than twice as large as that predicted for the deleted phosphodianion‐amide interaction. 2.) The effects of consecutive R269A and N270A mutations depend strongly on the order of these mutations. 3.) The N270A mutation results in a surprising 40‐fold increase in the second‐order rate constant for GPDH‐catalyzed reduction of GA, and causes HP i to change from a strong activator to a weak inhibitor of this GPDH‐catalyzed reaction. These large and complex changes in the activation barriers for hydride transfer show that the amide side chain of Asn270 functions as a “linchpin” in maintaining the structural integrity of the dianion activation site for GPDH.