| Pyruvate kinase (PK) is a key regulatory glycolytic enzyme. PK catalyzes the physiological phosphorylation of ADP by PEP. The active-site structure of PK showed that domain B of PK protrudes into the solvent and forms a cleft with domain A. Domain B is attached to domain A by what appears to be a flexible hinge region. The active site lies in the pocket between domains A and B of the same subunit. The inactive state can be represented by a rotation of the domain B in opening of the cleft between the B and A domains, and the active state can be represented by closing of the cleft. The domain B is highly mobile and differs in 40°in the two conformers, whereas the domain C remains in the same position in the "open" and "closed" conformations. The enzyme-metal-substrate complex has a conformation which is different from that of free enzyme. The UV and fluorescence specrtrum studies reveal that the solvating environment of certain protein chromophores is in a non-aqueous environment at high temperature or in the absence of cations, and in an aqueous environment at low temperature or in the presence of cations. The substrate, ADP, didn't perturb the UV spectrum of the protein in the presence of activating cations. Binding of PEP induces PK to assume a more compact and symmetric structure. In contrast, Phe loosens the protein structure into an inactive or less active state. The binding of activating cations and/or substrates or the inhibitor retains the relative distribution of the secondary structures.In this work, the conformational change of PK induced by its activating cations K+/Mg2+, substrates ADP/PEP, and inhibitor Phe, have been studied by using fluorescence acrylamide quenching. Isothermal titration calorimetry (ITC) was used to address the thermodynamic properties of the reaction between PK and activating cations and/or substrates. The results are as follows:1. There is no significant change in the secondary structure in PK induced by its activating matels K+/Mg2+, substrates ADP/PEP, inhibitor Phe. However, it involves domain movements and conformational changes. The active site of PK was brought into an aqueous environment by interactions of Mg2+ or K+/Mg2+. ADP has little contribution on the solvent accessibility of tryptophan residues. Binding of PEP or PEP/ADP elicits a decrease in accessibility of PK active site. Phe can inhibit the activity of PK and in contract substrate PEP, increase the solvent accessibility of PK tryptophan residues.2. When enzyme is heated from 10℃to 30℃, there is also no significant change in the secondary structure. However, temperature would change the conformation of PK active site, the solvent accessibility of active site is inversely related to temperature.3. As a homo-tetramer, PK can be dissociated into a less compact and in-active tetramer in the presence of 0.5M GdnHCl. In the presence of 1.5M GdnHCl, PK was dissociated into dimmers dimer with a partial loss of the secondary structure. PK is a disordered monomer in the presence of 2.5M GdnHCl. Noteworthy, activating cations and substrates can partially reverse the conformational change induced by GdnHCl.The conformational changes of Calcineurin (CN) induced by Ca2+/CaM binding was also studied in this work. CN is a calcium/CaM-dependent Ser/Thr protein phosphatase and plays a critical role in the coupling of Ca2+signals to cellular responses. CN is a heterodimeric enzyme consisting of a 61-kDa subunit (CNA) with catalytic activity and the binding sites for Ca2+/CaM and a 19-kDa subunit (CNB). The conformational change mechanism was studied by fluorescence acrylamide quenching. The results are:the isolated regulatory region (CNRR) inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The binding of Ca2+ to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. |
PDF Full Text Request