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

1994 ◽  
Vol 303 (1) ◽  
pp. 141-145 ◽  
Author(s):  
T K Harris ◽  
V L Davidson
Keyword(s):  
Paracoccus Denitrificans ◽  
Double Resonance ◽  
Pyrroloquinoline Quinone ◽  
State Theory ◽  
Methanol Dehydrogenase ◽  
Activation Entropy ◽  
Host Bacterium ◽  
Prosthetic Group ◽  
Electron Nuclear Double Resonance ◽  
Delta G

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.

scholarly journals Replacement of enzyme-bound calcium with strontium alters the kinetic properties of methanol dehydrogenase

1994 ◽  
Vol 300 (1) ◽  
pp. 175-182 ◽  
Author(s):  
T K Harris ◽  
V L Davidson
Keyword(s):  
Paracoccus Denitrificans ◽  
Competitive Inhibitor ◽  
Methanol Dehydrogenase ◽  
Native Enzyme ◽  
Kinetic Properties ◽  
Host Bacterium ◽  
Prosthetic Group ◽  
Dependent Activity ◽  
Cyanide Inhibition ◽  
Endogenous Activity

Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone (PQQ) prosthetic group. Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. MEDH, which was purified from these cells (Sr-MEDH), exhibited an increased absorption coefficient for the PQQ chromophore, and displayed certain kinetic properties which were different from those of native MEDH. Native MEDH exhibits an endogenous activity which is not stimulated by substrate and which is inhibited by cyanide. Sr-MEDH exhibited lower endogenous activity which was stimulated by substrate, and was much less sensitive to inhibition by cyanide. The Vmax. for the methanol-dependent activity of Sr-MEDH was 3-fold greater than that of the native enzyme, and the Ks for methanol was altered. Cyanide also acts as an obligatory activator and competitive inhibitor of methanol-dependent activity in native MEDH from P. denitrificans [Harris and Davidson (1993) Biochemistry 32, 4362-4368]. Sr-MEDH exhibited a similar K1 for cyanide inhibition of methanol-dependent activity, but the KA for cyanide activation of this activity was 17-fold greater than that for the native enzyme. The activation energy of Sr-MEDH was 13.4 kJ (3.2 kcal)/mol lower than that of the native enzyme. These data confirm and significantly extend the conclusions from genetic [Richardson and Anthony (1992) Biochem. J. 287, 709-715] and crystallographic [White, Boyd, Mathews, Xia, Dai, Zhang and Davidson (1993) Biochemistry 32, 12955-12958] studies that suggest an apparently unique role for Ca2+ in MEDH compared with other Ca(2+)-dependent proteins and enzymes.

scholarly journals Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion

1992 ◽  
Vol 287 (3) ◽  
pp. 709-715 ◽  
Author(s):  
I W Richardson ◽  
C Anthony
Keyword(s):  
Paracoccus Denitrificans ◽  
Molecular Size ◽  
Methanol Dehydrogenase ◽  
Single Atom ◽  
Methylobacterium Extorquens ◽  
Prosthetic Group ◽  
Low Concentrations ◽  
L Proteins ◽  
Group 2 ◽  
Mutant Forms

Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer. The role of Ca2+ was investigated using the MDH from Methylobacterium extorquens. This was shown to be similar to the MDH from Hyphomicrobium X in having 2 mol of prosthetic group (pyrroloquinoline quinine; PQQ) per mol of tetramer, the PQQ being predominantly in the semiquinone form. MDH isolated from the methanol oxidation mutants MoxA-, K- and L- contained no Ca2+. They were identical with the enzyme isolated from wild-type bacteria with respect to molecular size, subunit configuration, pI, N-terminal amino acid sequence and stability under denaturing conditions (low pH, high urea and high guanidinium chloride) and in the nature and content of the prosthetic group (2 mol of PQQ per mol of MDH). They differed in their lack of Ca2+, the oxidation state of the extracted PQQ (fully oxidized), absence of the semiquinone form of PQQ in the enzyme, reactivity with the suicide inhibitor cyclopropanol and absorption spectrum, which indicated that PQQ is bound differently from that in normal MDH. Incubation of MDH from the mutants in calcium salts led to irreversible time-dependent reconstitution of full activity concomitant with restoration of a spectrum corresponding to that of fully reduced normal MDH. It is concluded that Ca2+ in MDH is directly or indirectly involved in binding PQQ in the active site. The MoxA, K and L proteins may be involved in maintaining a high Ca2+ concentration in the periplasm. It is more likely, however, that they fill a ‘chaperone’ function, stabilizing a configuration of MDH which permits incorporation of low concentrations of Ca2+ into the protein.

The Microscopic Structure Of Shallow Donors In Silicon Carbide

1996 ◽  
Vol 442 ◽  
Author(s):  
J.-M. Spaeth ◽  
S. Greulich-Weber ◽  
M. März ◽  
E. N. Kalabukhova ◽  
S. N. Lukin
Keyword(s):  
High Temperature ◽  
Low Temperature ◽  
Temperature Dependence ◽  
Double Resonance ◽  
Epr Spectra ◽  
Electron Nuclear Double Resonance ◽  
Paramagnetic Resonance ◽  
Vacancy Pair ◽  
Temperature Spectra ◽  
Electron Paramagnetic

AbstractThe electronic structure of nitrogen donors in 6H-, 4H- and 3C-SiC is investigated by measuring the nitrogen hyperfine (hf) interactions with electron nuclear double resonance (ENDOR) and the temperature dependence of the hf split electron paramagnetic resonance (EPR) spectra. Superhyperfine (shf) interactions with many shells of 13C and 29Si were measured in 6H-SiC. The hf and shf interactions are discussed in the framework of effective mass theory. The temperature dependence is explained with the thermal occupation of the lowest valley-orbit split A1 and E states. It is proposed that the EPR spectra of P donors observed previously in neutron transmuted 6H-SiC at low temperature (<10K) and high temperature (>60K) are all due to substitutional P donors on the two quasi-cubic and hexagonal Si sites, whereby at low temperature the E state is occupied and at high temperature the A1 state. The low temperature spectra are thus thought not to be due to P-vacancy pair defects as proposed previously.

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