title: Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis.
abstract: The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) 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 Mn(2+) 170-fold. In contrast, Asp(75) 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 Mn(2+) 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 Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.
authors.affiliation: Virginia Bioinformatics Institute and Departments of Biochemistry and Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Case
authors.forename: Edward M
authors.initials: EM
authors.lastname: Concar
authors.forename: Kristin L
authors.initials: KL
authors.lastname: Boswell
authors.forename: Biswarup
authors.initials: B
authors.lastname: Mukhopadhyay
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D003600:Cytosol / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D062907:Oxaloacetic Acid / Q000737:chemistry; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D062907:Oxaloacetic Acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D010729:Phosphoenolpyruvate Carboxykinase (GTP)