The complete nucleotide sequence of the carbonic anhydrase gene from Neisseria gonorrhoeae has been determined. The gene encodes a 252-residue polypeptide with a molecular mass of 28085 Da. The gene has been cloned and overexpressed in Escherichia coli, and the enzyme has been purified. A 26-residue signal peptide is cleaved off by the E. coli processing machinery. Thus, the isolated enzyme contains 226 amino acid residues with a molecular mass of 25 314 Da. Most of the enzyme seems to be produced as a soluble protein located in the periplasm of E. coli. The enzyme is homologous to carbonic anhydrases from the animal kingdom; it is an a-carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and I1 suggests that the secondary structures are essentially intact in the bacterial enzyme but that several loops are much shorter than in the human forms. Most of the active-site residues are identical to those found in the high-activity human isozyme 11. The bacterial enzyme has a high CO, hydration activity with a k,,, of 1.1 . lo6 s-' and K,, of 20 mM at pH 9 and 25°C.The enzyme also catalyzes the hydrolysis of 4-nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pK, of 6.7 and a maximal value of the catalytic second-order rate constant, k,,,,,

The metabolic reduction of 11-keto groups in glucocorticoid steroids such as cortisone leads to the nuclear receptor ligand cortisol. This conversion is an example of pre-receptor regulation and constitutes a novel pharmacological target for the treatment of metabolic disorders such as insulin resistance and possibly other derangements observed in the metabolic syndrome, such as hyperlipidemia, hypertension, and lowered insulinsecretion.ThisreactioniscarriedoutbytheNADPHdependent type 1 11␤-hydroxysteroid dehydrogenase (11␤-HSD1), an enzyme attached through an integral N-terminal transmembrane helix to the lipid bilayer and located with its active site within the lumen of the endoplasmic reticulum. Here we report the crystal structure of recombinant guinea pig 11␤-HSD1. This variant was determined in complex with NADP at 2.5 Å resolution and crystallized in the presence of detergent and guanidinium hydrochloride. The overall structure of guinea pig 11␤-HSD1 shows a clear relationship to other members of the superfamily of short-chain dehydrogenases/reductases but harbors a unique C-terminal helical segment that fulfills three essential functions and accordingly is involved in subunit interactions, contributes to active site architecture, and is necessary for lipid-membrane interactions. The structure provides a model for enzyme-lipid bilayer interactions and suggests a funneling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site.

Interconversion between cortisone and the glucocorticoid receptor ligand cortisol is carried out by 11beta-hydroxysteroid dehydrogenase (11beta-HSD)isozymes and constitutes a medically important example of pre-receptor control of steroid hormones. The enzyme 11beta-HSD type 1 (11beta-HSD1) catalyzes the conversion of cortisone to its active receptor-binding derivative cortisol, whereas 11beta-HSD type 2 performs the reverse reaction. Specific inhibitors against the type 1 enzyme lower intracellular levels of glucocorticoid hormone, with an important clinical application in insulin resistance and other metabolic disorders. We report here on the in vitro oxysterol-metabolizing properties of human and rodent 11beta-HSD1. The enzyme, either as full-length, membrane-attached, or as a transmembrane domain-deleted, soluble form, mediates exclusively conversion between 7-ketocholesterol and 7beta-hydroxycholesterol with similar k(cat) values as observed with glucocorticoid hormones. Thus, human, rat, and mouse 11beta-HSD1 have dual enzyme activities like the recently described 7alpha-hydroxysteroid dehydrogenase/11beta-hydroxysteroid dehydrogenase from hamster liver, but differ fundamentally from the latter in that 7beta-OH rather than 7alpha-OH dehydrogenase constitutes the second activity. These results demonstrate an enzymatic origin of species differences in 7-oxysterol metabolism, establish the origin of endogenous 7beta-OH cholesterol in humans, and point to a possible involvement of 11beta-HSD1 in atherosclerosis.

A murine carbonic anhydrase-related protein (CARP) has been expressed in Escherichia coli and purified to near homogeneity. The polypeptide chain consists of 290 amino acid residues and has a calculated molecular mass of 32 950 Da. By introducing two mutations, Arg 117 --*His and Ginll5---~ Gin, we created a metal-binding center homologous to that in the carbonic anhydrases from the animal kingdom. In contrast to unmodified CARP, this double mutant was isolated as a 1 : 1 zinc-protein complex. While unmodified CARP is catalytically inactive, the mutant catalyzes CO2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III. The activity is strongly inhibited by the powerful and selective carbonic anhydrase inhibitor, acetazolamide.

Rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetate and 4-nitrophenyl propionate catalyzed by wild-type and mutant forms of human carbonic anhydrase II have been measured. The results show that the mutations Tyr73Phe and Ala653Leu lead to activity enhancements with all the investigated substrates, but there is no significant effect on the specificity. In contrast, some mutations at sequence position 200 have large effects on specificity. For example, while the mutation Thr2003Gly results in a threefold increase of the rate of hydrolysis of 4-nitrophenyl acetate, the activity is enhanced 10 times with the meta-substituted substrate and 380 times with the orthosubstituted substrate. These results are interpreted in terms of the removal in the mutant of a steric interference between the 2-NO 2 group, in particular, and the side chain of Thr200. Mutants involving residues lining a hydrophobic pocket near the catalytically essential zinc ion have also been investigated. The most pronounced effect on specificity was found for the Val1433Gly mutant. This mutation leads to a sixfold decrease of the rate of hydrolysis of 4-nitrophenyl acetate but a 20-fold increase of the activity with the propionyl ester as substrate. These results suggest that the side chain of Val143 interferes sterically with the acyl moiety of 4-nitrophenyl propionate. Based on these results, we have constructed a hypothetical model of the location of these ester substrates in the enzymic active site.Keywords: carbonic anhydrase; esterase activity; mutagenesis; substrate specificity.The physiological reaction catalyzed by the zinc-containing enzyme carbonic anhydrase (CA, EC4.2.1.1) is the reversible hydration of carbon dioxide: CO 2 + H 2 O 6 HCO 3 2 + H + . According to the current mechanism model [1], the central catalytic step in CO 2 hydration is a nucleophilic attack of a zincbound OH 2 on a CO 2 molecule which is loosely bound in a hydrophobic pocket in the vicinity of the zinc ion. This step results in a zinc-coordinated bicarbonate ion which is subsequently displaced from the metal ion by a water molecule. The catalytic cycle is completed by the rate-limiting transfer of H + from this water molecule to the reaction medium.The enzyme can also act on other carbonyl systems, such as esters and aldehydes [2]. There is overwhelming evidence that catalysis of both ester hydrolysis and aldehyde hydration occur by a zinc-hydroxide mechanism. Thus, the pH dependence of these activities seems to be controlled by the ionization of the zinc-bound water molecule, and pH-rate profiles of CA-catalyzed hydrolysis of the chromogenic substrate, 4-nitrophenyl acetate, have been used routinely to estimate pK a values of zinc-bound H 2 O in various forms of the enzyme. Furthermore, mutations of active-site residues resulting in drastic losses of CO 2 hydration activity generally have severe effects on the esterase function as well. However, the molecular details of ester hydrolysis are not well understood, and the precise location of the ester substrate in...