Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis

Abstract

The roles of Asp75, Asp78, and Glu83 of the 75DPSDV ARVE 83 element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp78 and Glu83 are fully conserved in GTP-PEPCKs. 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 Km value for Mn2+ 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, Vmax decreased 1.4-4-fold. Km values for PEP and Mn2+ increased 3-9- and 1.2-10-fold, respectively. Km values for GDP and bicarbonate changed little. 2) For PEP formation, Vmax 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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.