Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract

Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis‐Menten kinetics for the substrates PEP, IDP, KHCO3, OAA and ITP with a Km of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

“…While microbial enzymes often use ATP as a phosphate donor, the C. glutamicum PEP carboxykinase has been shown to be highly specific for GTP (26)(27)(28)(29) and thus represents a notable exception. The kinetic analysis of the purified enzyme revealed ATP to inhibit PEP carboxykinase activity in the oxaloacetate-forming reaction (28), indicating that the enzyme mainly functions in gluconeogenesis and not in anaplerosis under physiological conditions.…”

“…While microbial enzymes often use ATP as a phosphate donor, the C. glutamicum PEP carboxykinase has been shown to be highly specific for GTP (26)(27)(28)(29) and thus represents a notable exception. The kinetic analysis of the purified enzyme revealed ATP to inhibit PEP carboxykinase activity in the oxaloacetate-forming reaction (28), indicating that the enzyme mainly functions in gluconeogenesis and not in anaplerosis under physiological conditions. Since PEP carboxykinase of C. glutamicum also shows significant activity in cells grown on glucose (23,28) and due to the fact that optimization of the cellular oxaloacetate concentration is crucial, especially for improved L-lysine produc-tion (30), deletion of the pck gene in a L-lysine-producing strain resulted in an increase in L-lysine productivity by 20% (23).…”

GntR-Type Transcriptional Regulator PckR Negatively Regulates the Expression of Phosphoenolpyruvate Carboxykinase in Corynebacterium glutamicum

“…The irreversible step of glycolysis that converts glucose-6-phosphate to PEP, i.e., the phosphofructokinase reaction, is bypassed by fructose-1,6-bisphosphatase. In contrast to all of the other bacterial PEP carboxykinases studied so far, the C. glutamicum enzyme shows high specificities for GTP and ITP instead of ATP; additionally, it is effectively inhibited by ATP (14). PEP carboxykinase is the only enzyme responsible for PEP synthesis, and it cannot be functionally replaced by the combined activities of malic enzyme or oxaloacetate decarboxylase and PEP synthetase (21).…”

Pyruvate:Quinone Oxidoreductase from Corynebacterium glutamicum : Purification and Biochemical Characterization

“…Due to its importance for the carbon flux distribution within the metabolism and for the precursor supply for amino acid synthesis, the phosphoenolpyruvate (PEP)-pyruvate node of this organism (see Fig. 1) has been intensively studied and much attention has been focused on some of the enzymes involved, e.g., pyruvate kinase, PEP carboxylase, pyruvate carboxylase, and PEP carboxykinase (24,33,34,51,52,55). The oxidative decarboxylation of pyruvate and thus the fueling of the tricarboxylic acid (TCA) cycle with acetyl coenzyme A (acetyl-CoA) in C. glutamicum have been generally attributed to the pyruvate dehydrogenase complex (16,40,61).…”