Characterization of a novel methanol dehydrogenase containing a Ba2+ ion at the active site

The quinoprotein methanol dehydrogenase (MDH) contains a Ca2+ ion at the active site. Ca2+-free enzyme (from a processing mutant) was used to obtain enzyme containing Sr2+ or Ba2+, the Ba2+-MDH being the first enzyme to be described in which a Ba2+ ion functions at the active site. The activation energy for oxidation of methanol by Ba2+-MDH is less than half that of the reaction catalysed by Ca2+-MDH (a difference of 21.4 kJ/mol), and the Vmax value is 2-fold higher. The affinities of Ba2+-MDH for substrate and activator are very much less than those of Ca2+-MDH; the Km for methanol is 3.5 mM (compared with 3 µM) and the KA for ammonia is 52 mM (compared with 2 mM). The different activity of Ba2+-MDH is probably due to a change in the conformation of the active site, leading to a decrease in the free energy of substrate binding and hence a decrease in activation energy. The kinetic model for Ba2+-MDH with respect to substrate and activator is consistent with previous models for Ca2+-MDH. The pronounced deuterium isotope effect (6.0–7.6) is influenced by ammonia, and is consistent with activation of the pyrroloquinoline quinone reduction step by ammonia. Because of its low affinity for substrates, it is possible to prepare the oxidized form of Ba2+-MDH. No spectral intermediates could be detected during reduction by added substrate, and so it is not possible to distinguish between those mechanisms involving covalent substrate addition and those involving only hydride transfer.

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The Effects of Increased Viscosity on the Function of Integral Membrane Proteins

High Pressure Effects in Molecular Biophysics and Enzymology ◽

10.1093/oso/9780195097221.003.0024 ◽

1996 ◽

Author(s):

Suzanne F. Scarlata

Keyword(s):

Activation Energy ◽

Membrane Proteins ◽

Active Site ◽

Protein Function ◽

Frequency Factor ◽

Integral Membrane Proteins ◽

Membrane Viscosity ◽

Internal Motions ◽

Soluble Enzymes

For many years the idea that the activity of integral membrane proteins is regulated by the fluidity of the lipid matrix was popular and appeared to be quite rational. However, as information about the effect of viscosity on the function of different membrane proteins became available, the correlation between the two became increasingly unclear. The purpose of this article is to readdress this issue in light of our recent pressure and temperature studies. This chapter is divided into seven parts: (1) the effect of viscosity on enzyme activity; (2) the effect of viscosity on the local motions of proteins; (3) characterization of membrane viscosity; (4) demonstration of changes in protein-lipid contacts brought about by changes in viscosity; (5) an example of a protein in which the viscosity appears to stabilize a particular conformational state: (6) relations between membrane viscosity and protein function; and (7) conclusions. The effect of viscosity (η) on the rate (k) of a chemical reaction was first given by Kramers (1940): . . . k=A/ηe−Ea/RT (1) . . . In this expression, viscosity will affect the rate of a reaction by limiting the rate of diffusion of reactants. Viscosity will thus modify the frequency factor (A) and should not affect the activation energy. This expression has been applied to aqueous soluble enzymes (for example, Gavish, 1979; Gavish & Werber, 1979; Somogyi et al., 1984), and it appears that, in general, enzymes obey Kramers’s relation, although in some cases the exponent of η is less than one. Viscosity can affect enzymatic rates not only by limiting the diffusion of substrates but also by damping internal motions of the protein chains. It seems reasonable that a high enough viscosities, the protein would be damped sufficiently so that large activation energies will be required for the backbone motions that allow substrates and products to diffuse into and out of the active site. This viscosity-induced increase in activation energy was shown by studies of the reassociation of carbon monoxide and dioxygen to the heme site of myoglobin after flash photodissociation (Austin et al., 1975; Beece et al., 1980).

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Pyrroloquinoline Quinone Biogenesis: Characterization of PqqC and Its H84N and H84A Active Site Variants†

Biochemistry ◽

10.1021/bi700162n ◽

2007 ◽

Vol 46 (24) ◽

pp. 7174-7186 ◽

Cited By ~ 16

Author(s):

Olafur Th. Magnusson ◽

Jordan M. RoseFigura ◽

Hirohide Toyama ◽

Robert Schwarzenbacher ◽

Judith P. Klinman

Keyword(s):

Active Site ◽

Pyrroloquinoline Quinone

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Characterization of the active site for the selective oxidation of methanol to formaldehyde on polycrystalline silver catalyst

Journal of the Chemical Society Chemical Communications ◽

10.1039/c39940001717 ◽

1994 ◽

pp. 1717 ◽

Cited By ~ 2

Author(s):

Graeme J. Millar ◽

James B. Metson ◽

Graham A. Bowmaker ◽

Ralph P. Cooney

Keyword(s):

Selective Oxidation ◽

Active Site ◽

Silver Catalyst ◽

Oxidation Of Methanol ◽

Polycrystalline Silver

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Trichoderma reesei Dehydrogenase, a Pyrroloquinoline Quinone-Dependent Member of Auxiliary Activity Family 12 of the Carbohydrate-Active Enzymes Database: Functional and Structural Characterization

Applied and Environmental Microbiology ◽

10.1128/aem.00964-19 ◽

2019 ◽

Vol 85 (24) ◽

Cited By ~ 5

Author(s):

Annick Turbe-Doan ◽

Eric Record ◽

Vincent Lombard ◽

Rajender Kumar ◽

Anthony Levasseur ◽

...

Keyword(s):

Trichoderma Reesei ◽

Structural Characterization ◽

Active Site ◽

Pyrroloquinoline Quinone ◽

Dimensional Structure ◽

Crystallographic Structure ◽

Coprinopsis Cinerea ◽

Carbohydrate Active Enzymes ◽

Content Type

ABSTRACT Pyrroloquinoline quinone (PQQ) is an ortho-quinone cofactor of several prokaryotic oxidases. Widely available in the diet and necessary for the correct growth of mice, PQQ has been suspected to be a vitamin for eukaryotes. However, no PQQ-dependent eukaryotic enzyme had been identified to use the PQQ until 2014, when a basidiomycete enzyme catalyzing saccharide dehydrogenation using PQQ as a cofactor was characterized and served to define auxiliary activity family 12 (AA12). Here we report the biochemical characterization of the AA12 enzyme encoded by the genome of the ascomycete Trichoderma reesei (TrAA12). Surprisingly, only weak activity against uncommon carbohydrates like l-fucose or d-arabinose was measured. The three-dimensional structure of TrAA12 reveals important similarities with bacterial soluble glucose dehydrogenases (sGDH). The enzymatic characterization and the structure solved in the presence of calcium confirm the importance of this ion in catalysis, as observed for sGDH. The structural characterization of TrAA12 was completed by modeling PQQ and l-fucose in the enzyme active site. Based on these results, the AA12 family of enzymes is likely to have a catalytic mechanism close to that of bacterial sGDH. IMPORTANCE Pyrroloquinoline quinone (PQQ) is an important cofactor synthesized by prokaryotes and involved in enzymatic alcohol and sugar oxidation. In eukaryotes, the benefit of PQQ as a vitamin has been suggested but never proved. Recently, the first eukaryotic enzyme using PQQ was characterized in the basidiomycete Coprinopsis cinerea, demonstrating that fungi are able to use PQQ as an enzyme cofactor. This discovery led to the classification of the fungal PQQ-dependent enzymes in auxiliary activity family 12 (AA12) of the Carbohydrate-Active Enzymes (CAZy) database (www.cazy.org) classification. In the present paper, we report on the characterization of the ascomycete AA12 enzyme from Trichoderma reesei (TrAA12). Our enzymatic and phylogenetic results show divergence with the only other member of the family characterized, that from the basidiomycete Coprinopsis cinerea. The crystallographic structure of TrAA12 shows similarities to the global active-site architecture of bacterial glucose dehydrogenases, suggesting a common evolution between the two families.

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Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661681 ◽

1986 ◽

Vol 56 (03) ◽

pp. 349-352 ◽

Cited By ~ 2

Author(s):

A Tripodi ◽

A Krachmalnicoff ◽

P M Mannucci

Keyword(s):

Venous Thromboembolism ◽

Active Site ◽

Inhibitory Activity ◽

Antithrombin Iii ◽

Factor Xa ◽

Molecular Defect ◽

Affinity Adsorption ◽

Italian Family ◽

Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

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