Lysine 213 and Histidine 233 Participate in Mn(II) Binding and Catalysis in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Abstract

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.

pH Dependence of the Reaction Catalyzed by Avian Mitochondrial Phosphoenolpyruvate Carboxykinase

“…This is also supported by mutagenic data from the ATP-dependent isozyme in yeast. In the experiments with yeast PEPCK, it was shown that mutation of the corresponding lysine and histidine residues resulted in an increase in the K D for Mn 2+ of 30-100-fold, respectively, and a decrease in k cat of 5000-fold (36). Further, it seems clear from the pH dependence on b and q that an additional water molecule (or proton) undergoes fast exchange at the metal site with increasing pH.…”

The Functional Glycosyltransferase Signature Sequence of the Human β1,3-Glucuronosyltransferase Is a XDD Motif

“…Altogether, these data show that divalent ions are essential for GlcAT-I activity as well as for substrate binding, but do not provide information on the activation mechanism. Kinetic analyses combined with site-directed mutagenesis have been successfully used to assess metal effects in enzyme activation (32,33). We thus initiated kinetic studies of Mn 2ϩ activation of wild-type and mutant GlcAT-I.…”

“…Although, our results favor a model where the metal interacts with the nucleotide sugar to form the enzyme substrate, given the complexity of metal enzyme activation mechanism per se, we presume that the GlcAT-1-Mn 2ϩ interactions may be more intricate. Thus, direct binding studies of Mn 2ϩ to the enzyme by methods such as electron paramagnetic resonance spectroscopy (32) or isothermal titration calorimetry (31) will be a step forward in the understanding of metal activation of GlcAT-I.…”

Nature of Protein–CO2 Interactions as Elucidated via Molecular Dynamics

“…Taken together, the data indicate that replacement of Lys 213 by Ala has a dramatic impact on the shape and flexibility of the protein backbone, particularly for the key CO 2 binding Arg 65 residue. Experimental site-directed mutagenesis studies [20][21][22]24 have revealed that Lys 213 in PEPCK is bound to Mn 2+ , implying that the side chain of Lys 213 probably does not bear the positive charge characteristic of protonated Lys; the microenvironment of Lys 213 is thought to be carefully balanced to allow this unusual protonation state, thereby enabling Mn 2+ binding without charge−charge repulsion. Disrupting this delicate environment by replacing Lys 213 with Ala, as in these simulations, has significant consequences on protein structure and apparently can lead to unusual behavior, such as the CO 2 pathway of Figure 6.…”

“…In addition to a crystal structure, there is a wealth of additional experimental data against which the present MD simulations can be compared, both to ensure the validity of the computational protocol and to clarify, verify, or extend experimental results. Experimentally studied PEPCK enzymes fall into both the ATP- and GTP-dependent categories and have been studied for species such as yeast, ,− rat, − and various bacteria. − ,,,− These diverse experimental studies have yielded valuable insight into the interactions of PEPCK with metal ions, substrates (both PEP and OAA), inhibitors, and nucleotides and have also elucidated the chemistry of phosphoryl transfer. However, the interaction of PEPCK with CO 2 has not been examined in nearly as much detail in these experiments.…”