A Direct Substrate–Substrate Interaction Found in the Kinase Domain of the Bifunctional Enzyme, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase

Abstract

To understand the molecular basis of a phosphoryl transfer reaction catalyzed by the 6-phosphofructo-2-kinase domain of the hypoxia-inducible bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), the crystal structures of PFKFB3AMPPCPfructose-6-phosphate and PFKFB3ADPphosphoenolpyruvate complexes were determined to 2.7 A and 2.25 A resolution, respectively. Kinetic studies on the wild-type and site-directed mutant proteins were carried out to confirm the structural observations. The experimentally varied liganding states in the active pocket cause no significant conformational changes. In the pseudo-substrate complex, a strong direct interaction between AMPPCP and fructose-6-phosphate (Fru-6-P) is found. By virtue of this direct substrate-substrate interaction, Fru-6-P is aligned with AMPPCP in an orientation and proximity most suitable for a direct transfer of the gamma-phosphate moiety to 2-OH of Fru-6-P. The three key atoms involved in the phosphoryl transfer, the beta,gamma-phosphate bridge oxygen atom, the gamma-phosphorus atom, and the 2-OH group are positioned in a single line, suggesting a direct phosphoryl transfer without formation of a phosphoenzyme intermediate. In addition, the distance between 2-OH and gamma-phosphorus allows the gamma-phosphate oxygen atoms to serve as a general base catalyst to induce an "associative" phosphoryl transfer mechanism. The site-directed mutant study and inhibition kinetics suggest that this reaction will be catalyzed most efficiently by the protein when the substrates bind to the active pocket in an ordered manner in which ATP binds first.

Structure-Based Development of Small Molecule PFKFB3 Inhibitors: A Framework for Potential Cancer Therapeutic Agents Targeting the Warburg Effect

“…An | F o |−| F c | omit map enabled unambiguous placement of N4A into theFru-6-P binding pocket of the kinase domain of PFKFB3 (Figure 6A). The N4A located at the Fru-6-P pocket could be superimposed onto the Fru-6-P modeled in the structure of PFKFB3 in a ternary complex with AMPPCP and Fru-6-P (PDB code: 2DWP) [29]. This structure provides clear evidence, supporting the kinetic observations that N4A competes with Fru-6-P for the same binding pocket in PFKFB3 (Figure 6B, C, D).…”

“…To facilitate this task, it was necessary to determine the molecular characteristics of N4A binding to PFKFB3 by crystallizing human PFKFB3 in the presence of N4A. We determined the structure of this complex to 2.4 Å resolution by a method of molecular replacement using the first PFKFB3 structure (PDB code: 2AXN) as a search model [29]. An | F o |−| F c | omit map enabled unambiguous placement of N4A into theFru-6-P binding pocket of the kinase domain of PFKFB3 (Figure 6A).…”

“…To determine steady-state initial reaction rates, the 2-Kase reactions were performed first and the Fru-2,6-BP produced was measured by a conventional enzyme-coupled kinetics assay as described previously [29], [36]. Initial rates of decrease in absorbance (Abs) at 340 nm were corrected with the rate of the control reaction in which no Fru-2,6-BP was present.…”

Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway

“…6), it is not unreasonable to suggest that methylation of these arginine residues contributes to constitutive PFKFB3 stabilization in cancer cells. Second, previous studies showed that R133, another arginine residue proximal to methylated arginine residues (R131/134) on the same helix, is able to interact with fructose-6-phosphate (F-6-P), the substrate of PFKFB3 with the covalent bond45. The current observation that R131/134K mutant-transfected cells displayed lower PFKFB3 activity than the WT-transfected cells (Fig.…”

Investigating combinatorial approaches in virtual screening on human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): A case study for small molecule kinases

“…Using ligands already known to bind to the F-6-P site from crystallographic evidence, namely, F-6-P [27] , F-2,6-P 2 [27] , EDTA [20] , and PEP [27] , a pharmacophore model was built and used to screen the NCI diversity set via MOE's pharmacophore screening module (Fig. 4).…”