Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 3. Mechanism

Abstract

Mammalian pyruvate kinase exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues. All of the isoforms, except the muscle isoform, exhibit typical allosteric behavior. The case of the muscle isoform is a conundrum. It is inhibited by an allosteric inhibitor, Phe, yet it has traditionally not been considered as an allosteric enzyme. In this series of study, an energetic landscape of rabbit muscle pyruvate kinase (RMPK) was established. The phenomenon of inhibition by Phe is shown to be physiological. Furthermore, the thermodynamics for the temperature fluctuation and concomitant pH change as a consequence of muscle activity were elucidated. We have shown that: 1. The differential number of protons released or absorbed with regards to the various linked reactions adds another level of control to shift the binding constants and equilibrium of active⇆inactive state changes; the latter controls quantitatively the activity of RMPK. 2. ADP plays a major role in the allosteric mechanism in RMPK under physiological temperatures. Depending on the temperature, ADP can assume duel and opposite roles of being an inhibitor by binding preferentially to the inactive form and a substrate. 3. Simulation of the RMPK behavior under physiological conditions shows that the net results of the twenty one thermodynamic parameters involved in the regulation are well tuned to allow maximal response of the enzyme to even minute changes of temperature and ligand concentrations. KeywordsTwo state model; allosterism; thermodynamic parameters; binding constants; predictions Pyruvate kinase, an important glycolytic enzyme, catalyzes a transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP. This process yields a molecule of pyruvate and ATP. The enzyme has been extensively reviewed by Kayne (1). Mammalian PK exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues (2, 3). All of the isoforms, except the muscle isoform, exhibit typical allosteric behavior. The case of the muscle isoform is a conundrum. It is inhibited by an allosteric inhibitor, Phe, yet it has traditionally not been considered as an allosteric enzyme because the concentration required to elicit demonstrable inhibitory effect is too high to be considered physiological. In this series of study the goal is to establish an energetic landscape of rabbit muscle pyruvate kinase (RMPK) in order to resolve this issue of whether the phenomenon of inhibition by Phe is physiological. Furthermore, there is a long standing interest in the temperature fluctuation and

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 1. Calorimetric Study

“…These conclusions are further strengthened by our fluorescence data and model simulations described in two following papers of this issue (36, 37). …”

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 2. Fluorescence Study

“…At 23 °C the resulting free energy change for the R→T transition can be estimated to be ~ 3.4 kcal/(mol of tetramer), decreasing to zero at 38 °C, which is close to a rabbit body temperature (36–38). Significance of this finding is discussed in the next paper (30). …”

“…2. Visual inspection of the figure indicates that the fluorescence quantum yield of the R-state is lower than that of the T-state as evident by a gradual increase of I PEP / I 0 at low temperatures and a sharp decrease above 30°C where the fraction of the unliganded T-state is known to significantly increase (1, 4, 30). Due to the preferential affinity of PEP for the R-state, the presence of PEP shifts the enzyme population toward the R-state resulting in a decrease of fluorescent intensity.…”

“…Importantly, we confirmed an increase of the isobaric heat capacity associated with the entropy driven R→T transition and an intrigue role of ADP in the enzyme activation process. A graphical representation of current results, their physiological relevance, and simulated predictions of the RMPK allosteric regulation is presented in the next paper (30). …”

Enhancing Thermal Exchange in Humans and Practical Applications

“…In vitro studies of rabbit skeletal muscle have shown that pyruvate kinase, a critical enzyme for the flow of substrate into oxidative phosphorylation, is highly temperature sensitive. [14][15][16] Its activity is greatest around 35°C-38°C and drops off rapidly as the temperature rises due to conformational changes. If the rise in muscle temperature during exercise compromises the activity of pyruvate kinase, then the flow of the substrate into the Krebs cycle and, therefore, the rate of production of ATP will be reduced.…”