Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Previous work has demonstrated that the enzyme cycles between a catalytically inactive open state and a catalytically active closed state. The transition of the enzyme between these states requires the transition of several active site loops to shift from mobile, disordered structural elements to stable ordered states. The mechanism by which these disorder-order transitions are coupled to the ligation state of the active site however is not fully understood. To further investigate the mechanisms by which the mobility of the active site loops is coupled to enzymatic function and the transitioning of the enzyme between the two conformational states, we have conducted structural and functional studies of point mutants of E89. E89 is a proposed key member of the interaction network of mobile elements as it resides in the R-loop region of the enzyme active site. These new data demonstrate the importance of the R-loop in coordinating interactions between substrates at the OAA/PEP binding site and the mobile R- and Ω-loop domains. In turn, the studies more generally demonstrate the mechanisms by which the intrinsic ligand binding energy can be utilized in catalysis to drive unfavorable conformational changes, changes that are subsequently required for both optimal catalytic activity and fidelity.

Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions

“…We explored the possibility that in response to high glucose, p300 acetylates PCK1 and alters its catalytic activity. Due to the broad kinetic and structural information of rat PCK1 (Cui et al, 2017; Johnson and Holyoak, 2010, 2012; Johnson et al, 2016), the rat enzyme was overexpressed and purified as a His-tagged protein from HEK293T cells exposed to different glucose concentrations (Figure S1A). PCK1 activity was measured under V max conditions (saturating levels of substrates) in both forward and reverse reaction directions and compared to the identical recombinant protein expressed and purified from E. coli , as a control.…”

“…Except for K524AcK, acetylation on Lys91, 473 and 521 turned the gluconeogenic (OAA → PEP) reaction unfavorable (Table S3). It is known that PCK1 exhibits pyruvate kinase (PK) activity that increases with mutations affecting the active-site structure (Johnson et al, 2016). We used this PK activity to probe alterations in the active site.…”

“…K91AcK showed higher (5-fold) PK activity (Table S3C and Figure 3D), as was also observed for K473AcK and K521AcK variants (Table S3C), indicating alterations of the catalytic site. We also assessed the ability of the acetylated variants to maintain the integrity of the enolate intermediate by performing the PK reaction in the absence of a phosphoryl donor (GDP instead of GTP) (Johnson and Holyoak, 2010; Johnson et al, 2016). With the altered active site, the PK reaction of PCK1 is blocked and the enolate is more easily transformed into pyruvate by spontaneous protonation.…”

Proteolytic cleavage ofArabidopsis thalianaphosphoenolpyruvate carboxykinase-1 modifies its allosteric regulation

“…OAA + ATP PEP + ADP + CO 2 . Although this reaction is fully reversible in vitro, it is generally accepted that it proceeds towards OAA decarboxylation in vivo (Johnson et al, 2016). PEPCK requires two divalent cations to catalyze the reaction: one Mn 2+ ion acts as an essential activating cofactor that promotes OAA decarboxylation and stabilizes the enolate ion during catalysis; while one Mg 2+ cation forms the metal nucleotide complex that constitutes the active form of the substrate (Goldie and Sanwal, 1980;Burnell, 1986;Matte et al, 1997;Johnson et al, 2016).…”

“…Although this reaction is fully reversible in vitro, it is generally accepted that it proceeds towards OAA decarboxylation in vivo (Johnson et al, 2016). PEPCK requires two divalent cations to catalyze the reaction: one Mn 2+ ion acts as an essential activating cofactor that promotes OAA decarboxylation and stabilizes the enolate ion during catalysis; while one Mg 2+ cation forms the metal nucleotide complex that constitutes the active form of the substrate (Goldie and Sanwal, 1980;Burnell, 1986;Matte et al, 1997;Johnson et al, 2016). ATP-dependent PEPCK has different physiological roles in plants: i) it is part of the CO 2 -concentrating mechanisms operating in C 4 and CAM photosynthesis (Edwards et al, 1971; Reiskind and Bowes, 1991;Martín et al, 2011); ii) it participates in biotic and abiotic stress responses (Saez-Vasquez et al, 1995;Chen et al, 2000Chen et al, , 2002Saito et al, 2008;Penfield et al, 2012;Choi et al, 2015); iii) it is involved in nitrogen and amino acid metabolism, especially during fruit development (Walker et al, 1999;Lea et al, 2001); and iv) it is involved in gluconeogenesis during seed germination, channeling carbon released from fatty acid reserves to form sugars, until the photosynthetic apparatus is fully developed (Rylott et al, 2003;Penfield et al, 2004;Malone et al, 2007;Graham, 2008;Eastmond et al, 2015).…”