Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens

Abstract

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of approximately 66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium.

First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…As seen in the case of other GTP-PCKs (5,14,20), ITP and IDP acted as alternative nucleotide cofactors with similar V max values and slightly higher K m values than those for GTP and GDP. ATP and ADP were poor cofactors for Pck Tk but gave relatively high V max values (10 to 30% of those with GTP and GDP) compared to those of classical GTP-PCKs (1,14,20,24). However, the K m values for ATP and ADP were much higher than those of their guanyl counterparts.…”

“…In the L-lysine producer Corynebacterium glutamicum, GTP-PCK in glucose-grown cells has been reported to be an enzyme responsible for the recycling of anaplerotically synthesized excess OAA to PEP (17,18). In other bacteria, as suggested for the GTP-PCK in the anaerobic ruminal bacterium Ruminococcus flaverfacience (24), PCKs have been found to fulfill an anaplerotic role when the tricarboxylic acid cycle is utilized for anabolic purposes, such as organic and amino acid synthesis.…”

A GTP-dependent Vertebrate-type Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis

“…It is the gluconeogenesis role that makes GTP-PCK a potential target in the treatment for noninsulin-dependent diabetes mellitus (10). In parasites and in certain bacteria, GTP-PCK fulfills an anaplerotic role by carboxylating PEP to OAA (2,3,11,12). For all of these reasons, this enzyme has been a focus of intense mechanistic studies since its discovery (1,9).…”

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

“…Consequently, in vivo it often catalyzes the formation of PEP from OAA, the first committed and rate-controlling step in gluconeogenesis and glycerogenesis (6). Only rarely will an organism use this enzyme for anaplerosis or OAA formation (12)(13)(14). Human liver, kidney, and small intestine use GTP-PEPCK activity for the production of glucose during fasting (15)(16)(17)(18).…”