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Journal of Biochemistry Advance Access originally published online on May 28, 2008
Journal of Biochemistry 2008 144(3):305-312; doi:10.1093/jb/mvn069
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© 2008 The Japanese Biochemical Society

Crystal Structure of Pyruvate Kinase from Geobacillus stearothermophilus

Kenichiro Suzuki, Sohei Ito, Akiko Shimizu-Ibuka and Hiroshi Sakai*

Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422-8526, Japan

*To whom correspondence should be addressed. Tel: +81-54-264-5576, Fax: +81-54-264-5099, E-mail: gp1314{at}mail.f.u-shizuoka-ken.ac.jp

Received March 14, 2008; Accepted April 21, 2008


  Abstract

The pyruvate kinase (PK) from a moderate thermophile, Geobacillus stearothermophilus, is an allosteric enzyme activated by AMP and ribose 5-phosphate but not fructose 1, 6-bisphosphate (FBP), which is a common activator of PKs. It has an extra C-terminal sequence (ECTS), which contains a highly conserved phosphoenolpyruvate (PEP) binding motif, but its function and structure remain unclear. To elucidate the structural characteristics of the effector-binding site and the ECTS, the crystal structure of the C9S/C268S mutant of the enzyme was determined at 2.4 Å resolution. The crystal belonged to space group P6222, with unit cell parameters a, b = 145.97 Å, c = 118.03 Å. The enzyme was a homotetramer and its overall domain structure was similar to the previously solved structures except that the ECTS formed a new domain (C' domain). The structure of the C' domain closely resembled that of the PEP binding domain of maize pyruvate phosphate dikinase. A sulphate ion was found in a pocket in the effector-binding C domain. This site corresponds to the 6-phosphate group-binding site in yeast PK bound FBP and seems to be the effector-binding site. Through comparison of the structure of the putative effector-binding site to that of the FBP binding site of the yeast enzyme, the structural basis of the effector specificity of the G. stearothermophilus PK is discussed.

Key Words: allosteric, AMP, crystal structure, pyruvate kinase, ribose 5-phosphate

Abbreviations: A0.5, activator concentration required for half maximal activation; ECTS, extra C-terminal sequence; FBP, fructose 1, 6-bisphosphate; GstPK, Geobacillus stearothermophilus pyruvate kinase; GstPKNC, Geobacillus stearothermophilus pyruvate kinase C9S/C268S mutant; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PPDK, maize pyruvate phosphate dikinase; R5P, ribose 5-phosphate; S0.5, substrate concentration required for half maximal saturation


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