Carbonic anhydrases kinetics

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... 

The carbonic anhydrase (Cam) in M. thermophila cells is elevated several fold when the energy source is shifted to acetate, suggesting a role for this enzyme in the acetate-fermentation pathway. It is proposed that Cam functions outside the cell membrane to convert CO2 to a charged species (reaction A4) thereby facilitating removal of product from the cytoplasm. Cam is the prototype of a new class (y) of carbonic anhydrases, independently evolved from the other two classes (a and P). The crystal structure of Cam reveals a novel left-handed parallel P-helix fold (Kisker et al. 1996). Apart from the histidines ligating zinc, the activesite residues of Cam have no recognizable analogs in the active sites of the a- and P-classes. Kinetic analyses establish that the enzyme has a zinc-hydroxide mechanism similar to that of Cab (Alber et al. 1999). 

Alber BE, Colangelo CM, Dong J, et al. 1999. Kinetic and spectroscopic characterization of the gamma carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila. Biochemistry 38 13119-28. 

Smith KS, Gosper NJ, Stalhandske C, et al. 2000. Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase. J Bacteriol 182 6605-13. 

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,... 

Importantly, carbonic anhydrase II is one of the most efficient biological catalysts known and it catalyzes the hydration of CO2 with a turnover rate of 10 sec at 25 C (Khalifah, 1971 Steiner et al, 1975). With kcaJKm = 1.5 X 10 sec carbonic anhydrase II is one of a handful of enzymes for which catalysis apparently approaches the limit of diffusion control. Since transfer of the product proton away from the enzyme to bulk solvent comprises a kinetic obstacle [an enzyme-bound group with ap/C, of about 7 cannot transfer a proton to bulk solvent at a rate faster than 10 sec (for a review see Eigen and Hammes, 1963)], the observed turnover rate of 10 sec" requires the participation of buffer in the proton transfer. 

The results of kinetic and X-ray crystallographic experiments on mutant carbonic anhydrases II, in which side-chain alterations have been made at the residue comprising the base of the hydrophobic pocket (Val-143), illuminate the role of this pocket in enzyme-substrate association. Site-specific mutants in which smaller hydrophobic amino acids such as glycine, or slightly larger hydrophobic residues such as leucine or isoleucine, are substituted for Val-143 do not exhibit an appreciable change in CO2 hydrase activity relative to the wild-type enzyme however, a substitution to the bulky aromatic side chain of phenylalanine diminishes activity by a factor of about 10 , and a substitution to tyrosine results in a protein which displays activity diminished by a factor of about 10 (Fierke et o/., 1991). 

The importance of the biochemistry of hydration of CO2 and dehydration of HCOg in an aqueous environment has led to extensive and invigorating research on the enzyme carbonic anhydrase pertaining to its structural details, metal ion cofactor, its coordination environment (12) and kinetic activity Model studies, both theoretical and experimental, have been undertaken using primarily the complexes of Zn(II), Mn(II), and Co(II), the latter one being its closest equivalent (13). 

It is the intention of the authors to present a brief account on metal carbonato complexes which have a direct bearing on the reversible hydration of CO2 by the enzyme carbonic anhydrase. Emphasis is placed on the integration of the kinetic and mechanistic concepts derived from the studies on model systems with the available kinetic, chemical and structural information on the enzyme carbonic anhydrase. To start, the kinetics and equilibria of dissolved CO2, relevant to the present context, are presented. 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

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). 

Kinetics of the carbonic anhydrase-catalyzed reversible hydration of C02 601... 

It should be noted that the kinetics for the human B isoenzyme are more complicated in that the pH dependence indicates that additional groups influence the rate. Studies with the isoenzyme carboxymethylated at His-200 prepared from 13C-labelled bromoacetate show that the pH dependence of the 13C NMR signal can be fitted to a curve with two pKa values of 6.0 and 9.2, but not to a curve with a single pKa. The second group could be the imidazole side-chain of His-200. Paramagnetically shifted 13C NMR resonances in the modified Co11 human carbonic anhydrase-B have been located by a novel method 498 This should allow the confirmation of an earlier postulate that the carboxymethyl carboxylate is a ligand for zinc in the modified enzyme. 

Some other reactions, such as aldehyde hydration (29) and e ter hydrolyses (30—33) are also catalyzed by the enzyme, but much j ess efficiently than the reversible hydration of CO 2. The esterase reaction, in particular, has been very useful in the kinetic analysis of carbonic anhydrase function, however. 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

Pocker, Y., and Guilbert, L. J. Carbonic anhydrase catalysed hydrolysis and decarboxylation. Kinetic studies of enzyme-catalysed decomposition of mono- and disubstituted derivatives of carbonic acid. Biochemistry 13, 70-78 (1974). 

Khalifah, R. G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J. Biol. Chem. 246, 2561-2573(1971). 

Taylor, P. W., and Burgen, A. S. V. Kinetics of carbonic anhydrase inhibitor complex formation. A comparison of anion- and sulfonamide-binding mechanisms. Biochemistry JO, 3859-3866(1971). 

Less than 10 years after the discovery of carbonic anhydrase in 1932, this enzyme was found to contain bound zinc, associated with catalytic activity. This discovery, remarkable at the time, made carbonic anhydrase the first known zinc-containing enzyme. At present, hundreds of enzymes are knovm to contain zinc. In fact, more than one-third of all enzymes either contain bound metal ions or require the addition of such ions for activity. The chemical reactivity of metal ions—associated with their positive charges, with their ability to form relatively strong yet kinetically labile bonds, and, in some cases, with their capacity to be stable in more than one oxidation state—explains why catalytic strategies that employ metal ions have been adopted throughout evolution. 

Figure 9.27. Kinetics of Water Deprotonation. The kinetics of deprotonation and protonation of the zinc-bound water molecule in carbonic anhydrase.

A study of spinach carbonic anhydrase showed very similar kinetic behavior, but also showed that the Zn is boimd to a sulfur atom. It was concluded that the two enzymes are convergently evolved, with different structures, but have equivalent functions. 

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