The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation

Meneely, Kathleen M.; McFarlane, Jeffrey S.; Wright, Collette L.; Vela, Kathryn; Swint-Kruse, Liskin; Fenton, Aron W.; Lamb, Audrey L.

doi:10.1016/j.abb.2023.109679

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation

Journal Article · Fri Jun 30 00:00:00 EDT 2023 · Archives of Biochemistry and Biophysics

DOI:https://doi.org/10.1016/j.abb.2023.109679· OSTI ID:2423941

Meneely, Kathleen M. ^[1]; McFarlane, Jeffrey S. ^[2]; Wright, Collette L. ^[2]; Vela, Kathryn ^[1]; Swint-Kruse, Liskin ^[3]; Fenton, Aron W. ^[3]; ^[1]

University of Texas, San Antonio, TX (United States)
University of Kansas, Lawrence, KS (United States)
University of Kansas Medical Center, Kansas City, KS (United States)

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated. The Entner-Doudoroff pathway does not have FBP as a pathway intermediate, and ZmPYK is not allosterically activated. In this work, we solved the 2.4 Å X-ray crystallographic structure of ZmPYK. The protein is dimeric in solution as determined by gel filtration chromatography, but crystallizes as a tetramer. The buried surface area of the ZmPYK tetramerization interface is significantly smaller than that of hlPYK, and yet tetramerization using the standard interfaces from higher organisms provides an accessible low energy crystallization pathway. Interestingly, the ZmPYK structure showed a phosphate ion in the analogous location to the 6-phosphate binding site of FBP in hlPYK. Circular Dichroism (CD) was used to measure melting temperatures of hlPYK and ZmPYK in the absence and presence of substrates and effectors. The only significant difference was an additional phase of small amplitude for the ZmPYK melting curves. We conclude that the phosphate ion plays neither a structural or allosteric role in ZmPYK under the conditions tested. In conclusion, we hypothesize that ZmPYK does not have sufficient protein stability for activity to be tuned by allosteric effectors as described for rheostat positions in the allosteric homologues.

View Accepted Manuscript (DOE)

Research Organization:: SLAC National Accelerator Laboratory, Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: American Heart Association Predoctoral Fellowship; National Institutes of General Medical Sciences; National Institutes of Health (NIH); National Institutes of Health Graduate Training Program in the Dynamic Aspects of Chemical Biology Grant; USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 2423941

Alternate ID(s):: OSTI ID: 1989277

Journal Information:: Archives of Biochemistry and Biophysics, Journal Name: Archives of Biochemistry and Biophysics Vol. 744; ISSN 0003-9861

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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