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J. Biol. Chem., Vol. 281, Issue 51, 39262-39272, December 22, 2006

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Roles of Asp⁷⁵, Asp⁷⁸, and Glu⁸³ of GTP-dependent Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis^*

Christopher L. Case¹, Edward M. Concar^¶2, Kristin L. Boswell, and Biswarup Mukhopadhyay^||3

From the Virginia Bioinformatics Institute and Departments of Biochemistry and ^||Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 and the ^¶Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

The roles of Asp⁷⁵, Asp⁷⁸, and Glu⁸³ of the ⁷⁵DPSDVARVE⁸³ element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp⁷⁸ and Glu⁸³ are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp⁷⁸ influences Tyr²²⁰; Tyr²²⁰ helps to position bound PEP, and Glu⁸³ interacts with Arg⁸¹. Experimental data on other PEPCKs indicate that Arg⁸¹ binds PEP, and the phosphate of PEP interacts with Mn²⁺ of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K_m value for Mn²⁺ 170-fold. In contrast, Asp⁷⁵ is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V_max decreased 1.4-4-fold. K_m values for PEP and Mn²⁺ increased 3-9- and 1.2-10-fold, respectively. K_m values for GDP and bicarbonate changed little. 2) For PEP formation, V_max increased 1.5-2.7-fold. K_m values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu⁸³-Arg⁸¹ interaction affected Arg⁸¹-PEP association. D78A change altered the Tyr²²⁰-PEP interaction. These events perturbed PEP-Mn²⁺ interaction and consequently affected catalysis severely. In contrast, substitutions at Asp⁷⁵, a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp⁷⁵ substitutions affected PEP-Mn²⁺ interaction by changing the positions of Asp⁷⁸, Arg⁸¹, and Glu⁸³, which translated to differential effects on two directions.

Received for publication, March 20, 2006 , and in revised form, October 2, 2006.

^* This work was supported in part by a start-up fund from the Virginia Bioinformatics Institute and a seed grant from the Institute for Biomedical and Public Health Sciences, Virginia Polytechnic and State University (to B. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a 2004 undergraduate summer research fellowship from the Fralin Biotechnology Center.

² Recipient of a Colgate-Palmolive undergraduate research award from the Biotechnology Center at the University of Illinois, Urbana-Champaign.

³ To whom correspondence should be addressed: Virginia Bioinformatics Institute, Bioinformatics I, Virginia Polytechnic Institute and State University, Washington St. 0477, Blacksburg, VA 24061. Tel.: 540-231-8015; Fax: 540-231-2606; E-mail: biswarup{at}vt.edu.

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