Enzymes protonation state

The enzyme exists in two different protonation states of the active site cysteines, each binding a different enantiomer. Conversion between enantiomers can be through the racemization path (upper manifold of Fig. 7.16) or through direct proton exchange with water (lower manifold of Fig. 7.16). Knowles and coworkers found that interconversion of enzyme protonation states was kinetically significant [82]. This was determined by measuring rates of tritiated proline washout as a function of the proline concentration. It was found that higher concentrations of proline promote slower washout of the Ca proton. Additional support for the rela-... 

The tripeptides investigated by Auld and Vallee (151) exhibit pH vs. cat/Xm profiles (Fig. 10 a) which indicate that the ionizations of two enzyme residues, apparent pKa s of 6.1 and 9.0, control catalytic activity. The plots shown in Figs. 10b and 10 c demonstrate that the low pH ionization affects only cat. while the high pH ionization affects only Am. These authors point out that the simplest scheme which is consistent with these data involves the existence of three enzyme protonation states (e.g., EH2, EH and E) which are important to the activity of carboxypeptidase A. The first ionization, EH25 EH-)-H+, generates the catalytically active from of the enzyme without altering the affinity of the enzyme for substrate.2)... 

The behaviour of the mutant enzymes where, for example, histidine-152 has been changed to alanine is compared with that of wild type enzymes.60 The 31P NMR chemical shift values and signal width for H152A mutant enzyme have shown the presence of two conformers open and closed forms of the enzyme that interconvert slowly on the NMR time scale. The tightness of the binding of the cofactor to the protein surface and its protonation state have been also discussed for intermediate Schiff bases in different steps of the catalytic cycle (Table 1). 

As in the case of enzymic catalysis, one can have one protonation state of the enzyme, say EH, that reacts with the affinity label and two other states, say and EH2+, that do not. 

A cyclohexadienyl Lewis adduct or salt formed by the reaction of a Lewis base with an aromatic compound. Such an adduct is apparently formed from the reaction of OH with 4-(A/-2-aminoethyl-2 -pyridyl disulfide)- -nitrobenzo-2-oxa-l,3-diazole (2PROD). 2PROD is a two-protonic-state electrophile used as a probe for enzyme active site nucleophiles and as a fluorescent re-... 

Lyophilized enzymes have a pH memory, meaning that the activity of the enzyme in organic solvent parallels its pH-activity profile of the aqueous solution from which it was lyophilized [36, 79-81]. However, very often acidic or basic mixtures within a nonaqueous reaction mixture such as reactant, products, or impurities, can disrupt this delicate protonation state, leading to changes in catalytic activity. To counteract this potential problem, solid-state buffers have been developed to protect the enzyme s protonation state in the nonaqueous environment [53, 82]. These solid-state buffers contain pairs of crystalline solids that can be intercon-... 

The effects of mutating surface charge are mimicked by changes of pH. Below pH 5, the carboxylates become protonated, whereas above pH 9 the ammonium groups of lysine residues deprotonate, causing them to lose their positive charge. The changes in protonation state cause perturbations in the titration curves of the enzyme at extremes of pH. These effects are most marked at low... 

A further question concerns the fate of the tritium in the second molecule of (5S)-[5-3H]ALA during the dehydratase reaction. The C-5 atom of the ALA molecules becomes the C-2 atom of PBG and loses one of its enantiotopic protons, while the ring is aromatized. When (5S)-[5-3H]ALA was reacted on the dehydratase and the product was isolated by careful chromatography on cellulose, the resulting PBG retained the tritium also at position 2 [81]. This means that the aromatization of the presumptive intermediate 71 takes place in an enzyme-bound state and involves specific abstraction of the 2-HRe atom (Fig. 39). 

Although manganese catalases have often been referred to as azide insensitive/ these enzymes actually are inhibited by azide and related molecules albeit at higher concentrations than are necessary for the heme enzymes. Penner-Hahn and co-workers (22) have shown that HN3 is the likely protonation state of the inhibitor and have calculated an apparent of 80 mM. Slope replots of the pH dependence of azide inhibition are linear with a slope of 1. These data can be used to calculate a true K of 300 mM. Because azide is a competitive inhibitor with respect to peroxide, it is likely that azide is bound directly to the manganese center. Recent EPR and lH paramagnetic relaxation enhancement studies support this viewpoint. Other inhibitors include fluoride and thiocyanide. All of the reported inhibition studies are consistent with the catalase cycle and hydroxylamine inhibition of the catalase cycle. 

The electron originally at Cua becomes equilibrated approx. equally between heme a and Cua with the same kinetics as the F state is formed (Figure 4). This electron is finally transferred to the binuclear site, together with uptake of another proton via the D-pathway, and the O state is formed in ca. 2 3 ms. Different forms of the O state that differ in spectroscopic and kinetic parameters have long been described in the literature, but the stmctural basis for these differences remains unclear. Most recently Verkhovsky et al. reported a distinct difference in function, where reduction of the enzyme in state O was coupled to proton translocation only when snch rednction followed immediately after oxidation of the rednced enzyme by O2. The structure of the binuclear... 

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