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Thermal stability of methanol dehydrogenase is altered by the replacement of enzyme-bound Ca2+ with Sr2+.

ABSTRACT: Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone prosthetic group. Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. At temperatures in the transition region for stability, the rate constants for inactivation of MEDH purified from these cells (Sr-MEDH) were 2-fold lower than those for MEDH. However, Arrhenius plots yielded an activation energy (Ea) of 699 kJ (167 kcal)/mol for MEDH compared with 640 kJ (153 kcal)/mol for Sr-MEDH. Further analysis by transition-state theory yielded values for the activation enthalpy (delta H*) and activation entropy (delta S*) of 696 kJ (166 kcal)/mol and 1.73 kJ (414 cal)/mol per K for MEDH and 637 kJ (152 kcal)/mol and 1.55 kJ (371 cal)/mol per K for Sr-MEDH. The higher rate of inactivation of MEDH than Sr-MEDH at higher temperatures is a consequence of a more favourable net gain in entropy. This positive entropy contribution increases at high temperatures, and reduces the more favourable stability obtained from the enthalpy contribution for the free energy (delta G*) of inactivation. The differences in these thermodynamic data are discussed in relation to the recently determined crystal structure of MEDH as well as 1H electron-nuclear double resonance studies of the influence of Sr2+ substitution on the structure of the pyrroloquinoline quinone-derived radical in MEDH.

SUBMITTER: Harris TK

PROVIDER: S-EPMC1137568 | biostudies-other | 1994 Oct

REPOSITORIES: biostudies-other

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Project description:The extracellular calcium-sensing receptor (CaSR) controls vital bone cell functions such as cell growth, differentiation and apoptosis. The binding of the native agonist (Ca2+) to CaSR activates the receptor, which undergoes structural changes that trigger a cascade of events along the cellular signaling pathways. Strontium (in the form of soluble salts) has been found to also be a CaSR agonist. The activation of the receptor by Sr2+ is considered to be the major mechanism through which strontium exerts its anti-osteoporosis effect, mostly in postmenopausal women. Strontium-activated CaSR initiates a series of signal transduction events resulting in both osteoclast apoptosis and osteoblast differentiation, thus strengthening the bone tissue. The intimate mechanism of Sr2+ activation of CaSR is still enigmatic. Herewith, by employing a combination of density functional theory (DFT) calculations and polarizable continuum model (PCM) computations, we have found that the Ca2+ binding sites 1, 3, and 4 in the activated CaSR, although possessing a different number and type of protein ligands, overall structure and charge state, are all selective for Ca2+ over Sr2+. The three binding sites, regardless of their structural differences, exhibit almost equal metal selectivity if they are flexible and have no geometrical constraints on the incoming Sr2+. In contrast to Ca2+ and Sr2+, Mg2+ constructs, when allowed to fully relax during the optimization process, adopt their stringent six-coordinated octahedral structure at the expense of detaching a one-backbone carbonyl ligand and shifting it to the second coordination layer of the metal. The binding of Mg2+ and Sr2+ to a rigid/inflexible calcium-designed binding pocket requires an additional energy penalty for the binding ion; however, the price for doing so (to be paid by Sr2+) is much less than that of Mg2+. The results obtained delineate the key factors controlling the competition between metal cations for the receptor and shed light on some aspects of strontium's therapeutic effects.

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Thermal stability of methanol dehydrogenase is altered by the replacement of enzyme-bound Ca2+ with Sr2+.

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