Impaired glutamatergic synaptic transmission in the PKU brain.

This paper reviews recent results of our investigation of the mechanisms whereby hyperphenylalaninemia may cause brain dysfunction in classical phenylketonuria (PKU). Acute applications of L-Phe in rat and mouse hippocampal and cerebrocortical cultured neurons, at a range of concentrations found in PKU brain, significantly and reversibly depressed glutamatergic synaptic transmission by a combination of pre- and postsynaptic actions: (1) competition for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptors; (2) attenuation of neurotransmitter release; (3) competition for the glutamate-binding site of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid and kainate (AMPA/kainate) receptors. Unlike L-Phe, its non-tyrosine metabolites, phenylacetic acid, phenylpyruvic acid, and phenyllactic acid, did not produce antiglutamatergic effects. L-Phe did not affect inhibitory gamma-aminobutyric (GABA)-ergic transmission. Consistent with this specific pattern of effects caused by L-Phe in neuronal cultures, the expression of NMDA receptor NR2A and AMPA receptor Glu1 and Glu2/3 subunits in brain of hyperphenylalaninemic PKU mice (Pah(enu2) strain) was significantly increased, whereas expression of the NMDA receptor NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Considering the important role of glutamatergic synaptic transmission in normal brain development and function, these L-Phe-induced changes in glutamatergic synaptic transmission in PKU brain may be a critical element of the neurological symptoms of PKU.

Long-term changes in glutamatergic synaptic transmission in phenylketonuria.

The cellular mechanisms that underlie impaired brain function during phenylketonuria (PKU), the most common biochemical cause of mental retardation in humans, remain unclear. Acute application of L-Phe at concentrations observed in the PKU brain depresses glutamatergic synaptic transmission but does not affect GABA receptor activity in cultured neurons. If these depressant effects of L-Phe take place in the PKU brain, then chronic impairment of the glutamate system, which may contribute to impaired brain function, could be detected as changes in postsynaptic glutamate receptors. This hypothesis was tested by using a combination of liquid chromatography-mass spectrometry, patch-clamp, radioligand binding and western blot approaches in forebrain tissue from heterozygous and homozygous (PKU) Pah(enu2) mice. Brain concentrations of L-Phe were nearly six-fold greater in PKU mice (863.12 +/- 17.96 micromol/kg) than in their heterozygous counterparts (149.32 +/- 10.23 micromol/kg). This concentration is significantly higher than the K(B) of 573 microM for L-Phe to compete for N-methyl-D-aspartate (NMDA) receptors. Receptor binding experiments with [3H]MK-801 showed significant up-regulation of NMDA receptor density in PKU mice. Consistent with the depressant effects of L-Phe, expression of NMDA receptor NR2A and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Glu1 and Glu2/3 subunits was significantly increased, whereas expression of the NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Given the role of the glutamatergic system in brain development and function, these changes may, at least in part, explain the brain disorders associated with PKU.

The catalytic properties of human carbonic anhydrase IX.

Human carbonic anhydrase IX (CA IX) is an integral membrane protein and a member of the alpha class of carbonic anhydrases that includes the human and animal enzymes. We have prepared a truncated, recombinant form of human CA IX of 255 residues consistent with full-length human CA II, among the most efficient of the carbonic anhydrases. Catalysis by and inhibition of this form of human CA IX has been investigated using stopped-flow spectrophotometry and 18O exchange measured by mass spectrometry. In kinetic constants for the hydration of CO2, CA IX closely resembled CA II with maximal proton transfer-dependent 18O exchange near 1 micros(-1) and kcat/Km near 55 microM(-1) x s(-1). Human CA IX was very strongly inhibited by three classic sulfonamides and cyanate, with inhibition constants that are close to those for CA II.

Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors.

Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate-early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57Bl/6 mice. Potentially therapeutic serum levels of hAAT (600 microg/ml) were achieved after portal vein injection of doses of 4 x 10(9) infectious units (IU), a 10-fold lower dose than that required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3 x 10(10) IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.

Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle.

We report here that the DNA-dependent protein kinase (DNA-PK) affects the molecular fate of the recombinant adeno-associated virus (rAAV) genome in skeletal muscle. rAAV-human alpha1-antitrypsin (rAAV-hAAT) vectors were delivered by intramuscular injection to either C57BL/6 (DNA-PKcs(+)) or C57BL/6-SCID [severe combined immunodeficient (SCID), DNA-PKcs(-)] mice. In both strains, high levels of transgene expression were sustained for up to 1 year after a single injection. Southern blot analysis showed that rAAV genomes persisted as linear episomes for more than 1 year in SCID mice, whereas only circular episomal forms were observed in the C57BL/6 strain. These results indicate that DNA-PK is involved in the formation of circular rAAV episomes.

Proton transfer to residues of basic pK(a) during catalysis by carbonic anhydrase.

The maximal velocity in the hydration of CO(2) catalyzed by the carbonic anhydrases in well-buffered solutions is limited by an intramolecular proton transfer from zinc-bound water to acceptor groups of the enzyme and hence to buffer in solution. Stopped-flow spectrophotometry was used to accumulate evidence that this maximal velocity is affected by residues of basic pK(a), near 8 to above 9, in catalysis of the hydration of CO(2) by carbonic anhydrases III, IV, V, and VII. A mutant of carbonic anhydrase II containing the replacement His-64-->Ala, which removes the prominent histidine proton shuttle (with pK(a) near 7), allows better observation of these basic groups. We suggest this feature of catalysis is general for the human and animal carbonic anhydrases and is due to residues of basic pK(a), predominantly lysines and tyrosines more distant from the zinc than His-64, that act as proton acceptors. These groups supplement the well-studied proton transfer from zinc-bound water to His-64 in the most efficient of the carbonic anhydrases, isozymes II, IV, and VII.

Introduction of histidine analogs leads to enhanced proton transfer in carbonic anhydrase V.

The rate-limiting step in the catalysis of the hydration of CO2 by carbonic anhydrase involves transfer of protons between zinc-bound water and solution. This proton transfer can be enhanced by proton shuttle residues within the active-site cavity of the enzyme. We have used chemical modulation to provide novel internal proton transfer groups that enhance catalysis by murine carbonic anhydrase V (mCA V). This approach involves the site-directed mutation of a targeted residue to a cysteine which is then subsequently reacted with an imidazole analog containing an appropriately positioned leaving group. Compounds examined include 4-bromoethylimidazole (4-BEI), 2-chloromethylimidazole (2-CMI), 4-chloromethylimidazole (4-CMI), and a triazole analog. Two sites in mCA V, Lys 91 and Tyr 131, located on the rim of the active-site cavity have been targeted for the introduction of these imidazole analogs. Modification of the introduced Cys 131 with 4-BEI and 4-CMI resulted in enhancements of up to threefold in catalytic activity. The pH profiles indicate the presence of a new proton shuttle residue of pKa near 5.8, consistent with the introduction of a functional proton transfer group into the active site. This is the first example of incorporation by chemical modification of an unnatural amino acid analog of histidine that can act as a proton shuttle in an enzyme.

Carbon dioxide transport by proteic and facilitated transport membranes.

Membrane separation of gases is governed by the permeability of each species across the membrane. The ratio of permeabilities yields the selectivity. Use of certain organic carriers in facilitated transport membranes and the CO2 converting enzyme carbonic anhydrase (CA) in proteic and facilitated transport membranes allows a dramatic increase in CO2 selectivity over other gases. CA has a low Km (9 mM), which we predicted would allow it to scavenge CO2 to very low partial pressures. Our goal was to determine if CA could remove CO2 from an environment at levels of 0.1% or less. Prior measurements of CO2 transport across thin supported liquid membranes showed that addition of CA enhanced CO2 flux by 3- to 100-fold. Proteic films use bifunctional reagents (e.g., glutaraldehyde) to cross-link the enzyme forming a gel. Bovine serum albumin (BSA) is often added for structural stability. Using such a preparation we examined the ability of proteic films to improve CO2 selectivity and to scavenge CO2 from a mixed gas stream. Proof-of-concept results, measured by mass spectrometry, showed a fivefold improvement in CO2 capture rate with maximal improvement at CO2 values of 1% partial pressure difference in the presence of 0 atm absolute difference. At 0.1% CO2 the membrane exhibited a 76% improvement over controls. At 0.3% CO2 the improvement is about threefold. CA proteic membranes exhibit selectivity for CO2 over oxygen and nitrogen in excess of three orders of magnitude. A CA-based proteic or facilitated transport membrane should readily achieve CO2 partial pressures of 0.05% under CELSS conditions. In addition to proteic membranes we are exploring direct immobilization of engineered CA to ultra-high-permeability teflon membranes. Site-directed mutagenesis was used to add functional groups while retaining full enzymatic activity. These results provide a basis for development of far more efficient CO2 capture proteic and facilitated transport membranes with increased selectivity to values closer to 100-fold at 1% CO2. The result will be CO2 selectivity at 0.1% on the order of 400-fold. These results exceed those obtained with other technologies.

The catalytic properties of murine carbonic anhydrase VII.

Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.

Properties of intramolecular proton transfer in carbonic anhydrase III.

We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.

Intramolecular proton transfer from multiple sites in catalysis by murine carbonic anhydrase V.

The hydration of CO2 catalyzed by carbonic anhydrase requires proton transfer from the zinc-bound water at the active site to solution for each cycle of catalysis. In the most efficient of the mammalian carbonic anhydrases, isozyme II, this transfer is facilitated by a proton shuttle residue, His 64. Murine carbonic anhydrase V (mCA V) has a sterically constrained tyrosine at the analogous position; it is not an effective proton shuttle, yet catalysis by this isozyme still achieves a maximal turnover in CO2 hydration of 3 x 10(5) s-1 at pH > 9. We have investigated the source of proton transfer in a truncated form of mCA V and identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contribute to proton transfer. Intramolecular proton-transfer rates between these shuttle groups and the zinc-bound water were estimated as the rate-determining step in kcat for hydration of CO2 measured by stopped-flow spectrophotometry and in the exchange of 18O between CO2 and water measured by mass spectrometry. Comparison of kcat in catalysis by Lys 91 and Tyr 131 and the corresponding double mutant showed a strong antagonistic interaction between these sites, suggesting a cooperative behavior in facilitating the proton-transfer step of catalysis. Replacing four potential proton shuttle residues produced a multiple mutant that had 10% of the catalytic turnover kcat of the wild type, suggesting that the main proton shuttles have been accounted for in mCA V. These replacements caused relatively small changes in kcat/Km for hydration, which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers; these measurements serve as a control indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.

Isolation and expression of murine carbonic anhydrase IV.

A 1193-bp cDNA containing the complete murine carbonic anhydrase IV coding sequence was isolated from a Balb/c kidney cDNA library. The entire coding sequence plus shorter segments was used in an Escherichia coli T7 expression vector system to produce four forms of murine CA IV, including (1) a protein representing the full-length coding sequence, (2) an amino-truncated protein lacking the 18 N-terminal amino acid plasma membrane targeting sequence, (3) a protein which lacked the plasma membrane targeting sequence and 26 C-terminal amino acids, and (4) a protein which lacked both 36 N-terminal residues (the plasma membrane targeting sequence plus 18 additional amino acids which included the first two cysteines) and 26 C-terminal residues. All four proteins were expressed as catalytically inactive inclusion bodies. After rapid dilution of washed, guanidine hydrochloride-denatured inclusion bodies into a glutathione-, l-arginine-containing renaturation buffer, an active carbonic anhydrase IV at yields of 3-4 mg/liter was easily purified from cultures expressing the form lacking the N-terminal targeting sequence and 26 C-terminal residues. The longest and shortest forms of carbonic anhydrase IV failed to refold into active enzyme under these conditions. The activity of purified recombinant carbonic anhydrase IV was highly resistant to sodium dodecyl sulfate, as is the native enzyme. This resistance presumably results from intramolecular disulfide bonds maintaining a functional active site configuration even in the presence of denaturing agents.

Catalytic properties of murine carbonic anhydrase IV.

A cDNA encoding the murine carbonic anhydrase IV (mCA IV) gene, modified to resemble a form of mature human carbonic anhydrase IV (Okuyama, T., Waheed, A., Kusumoto, W., Zhu, X. L., and Sly, W. S. (1995) Arch. Biochem. Biophys. 320, 315-322), was expressed in Escherichia coli. Inactive inclusion bodies were collected and refolded, and active enzyme was purified; the resulting mCA IV was used to characterize the catalysis of CO2 hydration using stopped flow spectrophotometry and 18O exchange between CO2 and water. Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for kcat for hydration of CO2 catalyzed by mCA IV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc-bound water molecule and solution. A role for His64 in transferring protons between the zinc-bound water and solution was confirmed by the 100-fold lower activity of the mutant of mCA IV containing the replacement His64 --> Ala. The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism. The maximal turnover number kcat for hydration of CO2 catalyzed by mCA IV was 1.1 x 10(6) s-1 at pH > 9. A pKa of 6.6 was estimated for the zinc-bound water molecule in mCA IV.

Glutamate and aspartate as proton shuttles in mutants of carbonic anhydrase.

Maximal turnover rates for the hydration of CO2 and the depletion of 18O from CO2 catalyzed by carbonic anhydrase III (CA III) and carbonic anhydrase V (CA V) are limited by proton transfer involving zinc-bound water or hydroxide in the active site. We have investigated the capacity of glutamic and aspartic acids at position 64 in human CA III and murine CA V to act as proton shuttles in this pathway. The distance from the Calpha of position 64 to the zinc is near 9.5 A in the crystal structures of both CA III and CA V. Rates of intramolecular proton transfer between these proton shuttle groups and the zinc-bound water molecule were estimated as the predominant rate-contributing step in the catalytic turnover kcat in the hydration of CO2 measured by stopped flow and in the 18O exchange between CO2 and water measured by mass spectrometry. We found that both glutamate and aspartate residues at position 64 are efficient proton shuttles in HCA III. The rate constant for intramolecular proton transfer from either residue to zinc-bound hydroxide is 4 x 10(4) s-1, about 20-fold greater than that of the wild type which has lysine at position 64. When the active site residue Phe 198 in human CA III was replaced with Leu, measurement of catalysis showed that Glu 64 retained but Asp 64 lost its capacity to act as a proton shuttle. These observations were supported in studies of catalysis by murine CA V which contains Leu 198; here again, Glu 64 acted as a proton shuttle, but Asp 64 did not. Phe 198 in HCA III is thus a significant factor in the capacity of the active site to sustain proton transfer, possibly through its stabilization of hydrogen-bonded water bridges that enhance proton translocation from both Glu and Asp at position 64 to the zinc-bound hydroxide.

Structure-based design of an intramolecular proton transfer site in murine carbonic anhydrase V.

Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the hydration of CO2 to produce bicarbonate and a proton. The catalytic properties of wild-type murine CA V suggest the presence of a proton shuttle residue having pKa = 9.2, the role of which is to transfer a proton from zinc-bound water to solution in the hydration direction to regenerate the zinc hydroxide form of the enzyme. Two likely candidates for shuttle residues are the tyrosines at positions 64 and 131 in the active site cavity. The crystal structure of wild-type carbonic anhydrase V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an orientation pointing away from the zinc by Phe 65, although Tyr 131 is oriented toward the zinc. We have prepared mutants of murine CA V replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the proton shuttle mechanism using stopped-flow spectrophotometry and the depletion of 18O from CO2 measured by mass spectrometry. Experiments with both single and double mutations showed that neither position 64 nor position 131 was a prominent site for proton transfer. However, a double mutant of CA V containing the two replacements, Tyr 64-->His and Phe 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to the altered catalytic properties, the crystal structure of the His 64/Ala 65 double mutant strongly suggested proton transfer by His 64 after removal of the steric hindrance of Phe 65. This is the first structure-based design of an efficient proton transfer site in an enzyme.

Structure determination of murine mitochondrial carbonic anhydrase V at 2.45-A resolution: implications for catalytic proton transfer and inhibitor design.

The three-dimensional structure of murine mitochondrial carbonic anhydrase V has been determined and refined at 2.45-A resolution (crystallographic R factor = 0.187). Significant structural differences unique to the active site of carbonic anhydrase V are responsible for differences in the mechanism of catalytic proton transfer as compared with other carbonic anhydrase isozymes. In the prototypical isozyme, carbonic anhydrase II, catalytic proton transfer occurs via the shuttle group His-64; carbonic anhydrase V has Tyr-64, which is not an efficient proton shuttle due in part to the bulky adjacent side chain of Phe-65. Based on analysis of the structure of carbonic anhydrase V, we speculate that Tyr-131 may participate in proton transfer due to its proximity to zinc-bound solvent, its solvent accessibility, and its electrostatic environment in the protein structure. Finally, the design of isozyme-specific inhibitors is discussed in view of the complex between carbonic anhydrase V and acetazolamide, a transition-state analogue. Such inhibitors may be physiologically important in the regulation of blood glucose levels.

Multiple deletions are detectable in mitochondrial DNA of aging mice.

Mutational damage to human mitochondrial DNA (mtDNA) can cause disorders in oxidative phosphorylation; speculation that such damage is involved in degenerative diseases and aging is common. We have detected deletions in mouse mtDNA which resemble those found in elderly humans or patients with certain mtDNA disorders. Five different mtDNA deletions, predicted from the positions of short, direct DNA repeats, were present in aged, but not young, mice. Deleted regions were surrounded by either exact or inexact repeats and occurred in both the major and minor regions of the mtDNA genome. The abundance of a particular deletion was generally related to the thermodynamic stability of the bounding repeat sequence. Deletions in aged mice were present at low levels (less than 0.01% of total mtDNA). However, in contrast to results from aged humans, deletions were more abundant in liver than in brain, heart, or skeletal muscle. These results make it possible to predict the location and relative abundance of deletions in any sequenced mtDNA, including inbred mouse strains differing in inherent natural lifespan. The inbred mouse model will allow a critical examination of the relationship between the presence and abundance of mtDNA deletions and the aging process.

Proton transfer by histidine 67 in site-directed mutants of human carbonic anhydrase III.

The ability of a histidine residue at position 67 in human carbonic anhydrase III to transfer protons in the catalytic pathway for the hydration of CO2 was investigated for a series of site-specific mutants. Wild-type carbonic anhydrase III has an arginine at this position with the C alpha of residue 67 about 9.4 A from the zinc. The active-site cavity contains no other residues capable of facile proton transfer. Rate constants for proton transfer from His 67 to the zinc-bound hydroxide were determined from the rate constants for the exchange of 18O between CO2 and water measured by mass spectrometry. A range of values for the pKa of zinc-bound water was achieved by replacement of phenylalanine with leucine and aspartate at position 198 adjacent to the zinc. Application of Marcus rate theory showed that intramolecular proton transfer involving His 67 had an intrinsic energy barrier of 1.3 +/- 0.3 kcal/mol and a thermodynamic work function for a preceding unfavorable equilibrium of 10.9 +/- 0.1 kcal/mol. We previously showed that proton transfer from histidine 64 in carbonic anhydrase III could be described by Marcus rate theory [Silverman, D. N., Tu, C. K., Chen, X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemistry 32, 10757-10762]. In comparison, proton transfer from His 67 must overcome a more unfavorable preceding equilibrium (a larger work function) that probably represents an energy requirement for proper alignment of donor and acceptor groups plus the intervening hydrogen-bonded water. Once this alignment is achieved, the intrinsic energy barrier appears the same for His 67 or His 64.

Catalytic properties of mouse carbonic anhydrase V.

A cDNA encoding the mouse carbonic anhydrase V gene was isolated by reverse transcription and polymerase chain reaction from BALB/c mouse liver mRNA. Vectors containing the full coding sequence as well as two different NH2-terminal truncated genes expressed enzymatically active protein in Escherichia coli. The carbonic anhydrase V produced by a vector containing the full coding sequence, which includes a possible NH2-terminal mitochondrial targeting signal, was proteolytically processed by E. coli and contained several amino-terminal ends. The two NH2-terminal truncated vectors deleted, respectively, 1) the 29-amino acid putative targeting sequence and 2) 51 amino acids, yielding a protein equivalent to a carbonic anhydrase (CA) V isolated from mouse liver mitochondria; and both vectors produced homogeneous protein fractions. These latter two forms of CA V had identical steady-state constants for the hydration of CO2, with maximal values of kcat/Km at 3 x 10(7) M-1 s-1 and kcat at 3 x 10(5) s-1 with an apparent pKa for catalysis of 7.4 determined from kcat/Km. In catalytic properties, mouse CA V is closest to CA I; however, in inhibition by acetazolamide, ethoxzolamide, and cyanate, CA V is very similar to CA II. Mouse CA V has a tyrosine at position 64, where the highly active isozyme II has histidine serving as a proton shuttle in the catalytic pathway. Investigation of a site-specific mutant of CA V containing the replacement Tyr64-->His showed that the unique kinetic properties of CA V are not due to the presence of tyrosine at position 64.