Question

6a. Many pumps are members of the P-type ATPases. If you discovered a new enzyme with a similar function, what reaction intermediate would help convince you that your enzyme was a member of this family?

6b. Describe the functional domains of the sarcoplasmic reticulum CA2+ ATPase.

Answer 1

Ans 6a . The P-type ATPases, also known as E1-E2 ATPases, are a large group of evolutionarily related ion and lipid pumps that are found in bacteria, archaea, and eukaryotes. P-type ATPases are α-helical bundle primary transporters named based upon their ability to catalyze auto- (or self-) phosphorylation (hence P) of a key conserved aspartate residue within the pump and their energy source, adenosine triphosphate (ATP). In addition, they all appear to interconvert between at least two different conformations, denoted by E1 and E2. P-type ATPases fall under the P-type ATPase (P-ATPase) Superfamily. In humans, P-type ATPases serve as a basis for nerve impulses, relaxation of muscles, secretion and absorption in the kidney, absorption of nutrient in the intestine and other physiological processes. Prominent examples of P-type ATPases are the sodium-potassium pump (Na+/K+-ATPase), the proton-potassium pump (H+/K+-ATPase), the calcium pump (Ca2+-ATPase) and the plasma membrane proton pump (H+-ATPase) of plants and fungi. So if you find new enzyme with similar function than you should go for checking ATP hydrolysis occurs in the cytoplasmic headpiece at the interface between domain N and P. Two Mg-ion sites form part of the active site. ATP hydrolysis is tightly coupled to translocation of the transported ligand(s) through the membrane and also check for these two popypeptides A unique feature of this member of Ptype ATPases is that catalytic activity and substrate transport are located on two different polypeptides.

Ans 6b The mechanism of ion pumping by Ca2+-ATPase appears fairly stochastic. In molecular dynamics simulations, we see that the bound Ca2+ in E1·2Ca2+ undergoes very large thermal fluctuations and continuous water attacks . Glu309 side chain appears to be the only obstacle for a water molecule to replace the Ca2+. Then, it is obvious that the occlusion requires the second seal that fixes the side chain of Glu309. In fact, a helix is employed for this purpose, not just a single residue, to cope with thermal fluctuations. That is the reason why the domain movements are so large and changes in domain interface are used to move the gating machinery. ATP, phosphate, Mg2+, Ca2+ and even water (and presumably protons also) are used as modifiers of the interfaces. Energy barriers between the principal intermediates appear to be comparable to the thermal energy, as the key events, e.g., the rotation of the A-domain, occur when ADP is released (or at least detached from the phosphorylation site). We can readily make E1P analogues simply by mixing the ATPase and stable phosphate analogues, such as AlF4−, and they even change spontaneously to E2P analogues when the Ca2+ concentrations are low [39]. That will be the reason why nearly 100% efficiency of energy utilisation is possible and ATP can be synthesised by backward reaction (e.g. [40]). Postulated a question on countertransport as “why countertransport of H+ is necessary despite that the SR membrane is leaky to H+”, and our answer was that “the charge imbalance caused by Ca2+ release has to be compensated at least in part by protonation”