Carbonic anhydrases constants

At 20 C, the rate constant for this uncatalyzed reaction, uncat is 0.03/sec. In the presence of the enzyme carbonic anhydrase, the rate constant for this reaction, is 10 /sec. 

CO3 species was formed and the X-ray structure solved. It is thought that the carbonate species forms on reaction with water, which was problematic in the selected strategy, as water was produced in the formation of the dialkyl carbonates. Other problems included compound solubility and the stability of the monoalkyl carbonate complex. Van Eldik and co-workers also carried out a detailed kinetic study of the hydration of carbon dioxide and the dehydration of bicarbonate both in the presence and absence of the zinc complex of 1,5,9-triazacyclododecane (12[ane]N3). The zinc hydroxo form is shown to catalyze the hydration reaction and only the aquo complex catalyzes the dehydration of bicarbonate. Kinetic data including second order rate constants were discussed in reference to other model systems and the enzyme carbonic anhy-drase.459 The zinc complex of the tetraamine 1,4,7,10-tetraazacyclododecane (cyclen) was also studied as a catalyst for these reactions in aqueous solution and comparison of activity suggests formation of a bidentate bicarbonate intermediate inhibits the catalytic activity. Van Eldik concludes that a unidentate bicarbonate intermediate is most likely to the active species in the enzyme carbonic anhydrase.460... 

As an example, consider an early calculation of isotope effects on enzyme kinetics by Hwang and Warshel [31]. This study examines isotope effects on the catalytic reaction of carbonic anhydrase. The expected rate-limiting step is a proton transfer reaction from a zinc-bound water molecule to a neighboring water. The TST expression for the rate constant k is... 

This reaction is essential in maintaining a constant pH in blood by the bicarbonate buffer system. Carbonic anhydrase, which contains a single zinc atom in its structure, has a molecular weight of about 30,000. In this structure, zinc is surrounded tetrahedrally by three histidine molecules and one water molecule. The exact role of the catalyst is not known, but it is believed to involve hydrolysis that can be represented as... 

Given the reaction and the very high stability constants involved, the production of cobalt carbonic anhydrase would require a solution not of ACS-grade cobalt nitrate but a 99.999999999999. .. 999% pure cobalt nitrate solution. What happened in the lab synthesis was that trace metals in the ACS-grade salt were selectively bound to the apo-carbonic anhydrase because their stability constant advantage was orders of magnitude over that of cobalt. The sample used to discover this was sub-milligram in mass. 

Fig. 1.14 (a) The pH dependence of the rate constants for the association of carbonic anhydrase B (E) and p-nitrobenzenesnlfonamide (S). The reaction is monitored by nsing stopped-flow and the quenching of a tryptophan fluorescence in the protein which occurs when sulfonamides bind. The full line fits Eqn. (1.226) with k = 3.5 X lO M- s-, pA g = 7.5 and pK =... 

Aromatic sulfonamides are specific inhibitors of carbonic anhydrase (E). The apparent second-order rate constants for association of p-nitrobenzenesulfonamide with (a) carbonic anhydrase-B and (b) the carboxymethylated derivative of the enzyme are shown against pH in Figure 1.14. Estimate using equations (1.225) and (1.226) the values for pAig, and k and he two possible schemes shown for both carbonic... 

The buried Cys-212 of human carbonic anhydrase B (3 pM) is virtually unreactive towards 2-chloromercuric-4-nitrophenol (60 pM) at pH 9.2, but upon the addition of only 40 pM CN , the half-life drops to 10 minutes which is an, at least, 75-fold rate enhancement. On first analysis, this would suggest that inhibitor binding to the enzyme has produced a conformational change or altered the — SH environment of the Cys—212. This is unexpected. How would you prove by kinetic experiments that the CN is binding to the mercury compound and not the enzyme and that this is changing the reactivity. The rate reaches a constant value at high [CN ]. 

For reactions in which one or more reactants or products is a gas, manometry (the measurement of pressure differences) can provide a convenient means for monitoring the course and kinetics of the reaction Thus, enzymes that can be assayed with this method include oxidases, urease, carbonic anhydrase, hydrogenase, and decarboxylases. For example, bacterial glutamate decarboxylase is readily assayed by utilizing a Warburg flask and measuring the volume of gas evolved at different times using a constant-pressure respirometer. ... 

LZ Avila, Y-H Chu, EC Blossey, GM Whitesides. Use of affinity capillary electrophoresis to determine kinetic and equilibrium constants for binding of arylsulfonamides to bovine carbonic anhydrase. J Med Chem 36 126-133,... 

Fig. 7 Mobility-shift assay for the determination of dissociation constant of the complex between anti-DNP rat monoclonal IgG21) antibody and charged ligands that contained the A-dinitrophenyl group. Mesityl oxide (MO) served as EOF marker, bovine carbonic anhydrase (CAB) and bovine a-lactalbumin (LA) as internal references. The DNP ligands with a charge of —1 (A) und —9 (B), respectively, were used as additives to the running buffer. (Reprinted with permission from Ref. 30. Copyright 1995 American Chemical Society.)...

These constants are similar to those reported for the structurally similar carbonic anhydrase inhibitor, dorzolamide hydrochloride, (6.4 and 8.2,... 

Thus although a protein can be made very specific for a particular metal, if it has a fold that generates matching properties to those possessed by the cation (e.g., carbonic anhydrase), this degree of matching is not required for the removal of heavy metals as they have such high binding constants to unconstrained sites. Clearly, there is a competition between two types of site for two metal ions ... 

The importance of maintaining the active site water network in CA II for efficient proton transfer was investigated by substituting different amino acids of varying size at position 65 and measuring the rate constants for proton transfer in the variant carbonic anhydrases... 

Another simple addition reaction is the hydration of C02 to form the bicarbonate ion. Without catalysis the reaction may require several seconds,4 5 the apparent first-order rate constant being -0.03 s 1 at 25°C. Cells must often hasten die process. The specific catalyst carbonic anhydrase is widespread in its distribution... 

In the Briggs-Maldane mechanism, when k2 is much greater than k-i, kcJKM is equal to kx, the rate constant for the association of enzyme and substrate. It is shown in Chapter 4 that association rate constants should be on the order of 108 s l M l. This leads to a diagnostic test for the Briggs-Haldane mechanism the value of kaJKu is about 107 to 108 s-1 M-1. Catalase, acetylcholinesterase, carbonic anhydrase, crotonase, fumarase, and triosephosphate isomerase all exhibit Briggs-Haldane kinetics by this criterion (see Chapter 4, Table 4.4). 

We can use the two hypothetical steps of section Clb i.e., that kcJKM be maximized and that KM be greater than [S], to set up criteria for judging the state of evolution of an enzyme whose function is to maximize rate. We recall from Chapter 3 that the maximum value of kcJKM is the rate constant for the diffusion-controlled encounter of the enzyme and substrate, and from Chapter 4 that this is about 108 to 109 s "1 M l. A perfectly evolved enzyme should have a kcJKM in the range of 108 to 109 s"1 and a KM greater than [S]. Using the data for kcJKM listed in Table 4.4 and the substrate concentrations and KM values mentioned in this chapter, it appears that carbonic anhydrase and triosephosphate isomerase are perfectly evolved for the maximization of rate, which agrees with the conclusions of W. J. Albery and J. R. Knowles on triosephosphate isomerase.5... 

It was once thought that the rate of equilibrium of the catalytic acid and basic groups on an enzyme with the solvent limited the rates of acid- and base-catalyzed reactions to turnover numbers of 103 s 1 or less. This is because the rate constants for the transfer of a proton from the imidazolium ion to water and from water to imidazole are about 2 X 103 s 1. However, protons are transferred between imidazole or imidazolium ion and buffer species in solution with rate constants that are many times higher than this. For example, the rate constants with ATP, which has a pKa similar to imidazole s, are about I0 J s 1 M-1, and the ATP concentration is about 2 mM in the cell. Similarly, several other metabolites that are present at millimolar concentrations have acidic and basic groups that allow catalytic groups on an enzyme to equilibrate with the solvent at 107 to 108 s-1 or faster. Enzyme turnover numbers are usually considerably lower than this, in the range of 10 to 103 s-1, although carbonic anhydrase and catalase have turnover numbers of 106 and 4 X 107 s 1, respectively. 

The calculated [using a quantized classical path (QCP) approach] and observed isotope effects and rate constants are in good agreement for the proton-transfer step in the catalytic reaction of carbonic anhydrase. This approach takes account of the role of quantum mechanical nuclear motions in enzyme reactions.208... 

Carbonic anhydrase is inhibited by monovalent anions. For the bovine enzyme, apparent binding constants range from about 1 M-1 for F to 5 x 105 M 1 for HS at 25° C and pH 7.5 (Table 4). The interaction... 

Table 7. Apparent stability constants for metal binding in procarboxypeptidase A, carboxypeptidase A and human carbonic anhydrase B...

A simple calculation reveals that the picture cannot be quite as simple. Carbonic anhydrase has an exceptionally high overall rate of reaction, its turnover number kcat is -5 x 105 s-1 consequently, the rate constants of individual steps must be greater than this number. The acid dissociation of a Zn11 aqua species seems to be inconsistent with this requirement. The dissociation constant fQ can be written as the ratio of forward k and backward kh rate constants [Eq. (9.20)]. 

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