Chingkuang Tu

Catalytic Properties of Human Manganese Superoxide Dismutase

The depletion of superoxide catalyzed by human manganese superoxide dismutase (MnSOD) was observed spectrophotometrically by measuring the absorbance of superoxide at 250-280 nm following pulse radiolysis and by stopped-flow spectrophotometry. Catalysis showed an initial burst of activity lasting approximately 1 ms followed by the rapid emergence of a greatly inhibited catalysis of zero-order rate. These catalytic properties of human MnSOD are qualitatively similar to those reported for MnSOD from Thermus thermophilus (Bull, C., Niederhoffer, E. C., Yoshida, T., and Fee, J. A. (1991) J. Am. Chem. Soc. 113, 4069-4076). However, there are significant quantitative differences; the emergence of the inhibited form is approximately 30-fold more rapid for human MnSOD. The turnover number for human MnSOD at pH 9.4 and 20°C was kcat = 4 × 104 s−1 and kcat/Km = 8 × 108 M−1 s−1, determined by a simulated fit of the model of Bull et al. (1991) to the pulse radiolysis data. We also report that the maximum of the visible absorption spectrum of human MnSOD (ε480 = 525 M−1 cm−1) showed a strong dependence on pH that could be described by an ionization of pKa 9.4 ± 0.1 with a maximum at low pH.

Characterization of the Product-inhibited Complex in Catalysis by Human Manganese Superoxide Dismutase

The reduction with excess H2O2 of human Mn(III) superoxide dismutase (SOD) and the active-site mutant Y34F Mn(III)SOD was measured by scanning stopped-flow spectrophotometry and revealed the presence of an intermediate in the reduction of the manganese. The visible absorption spectrum of this intermediate closely resembled that of the enzyme in the inhibited, zero-order phase of the catalyzed disproportionation of superoxide. The decay of the visible spectrum of this intermediate was 2-fold faster for the wild-type compared with the mutant Y34F Mn-SOD. This correlates with the enhanced product inhibition of Y34F during the catalysis of O⨪2 dismutation. The visible spectrum of the product-inhibited complex resembles that of the azide-Mn-SOD complex, suggesting that the inhibited complex has expanded geometry about the metal to octahedral. This study shows that the inhibited complex responsible for the zero-order phase in the catalysis by Mn-SOD of superoxide dismutation can be reached through both the forward (O⨪2) and reverse (H2O2) reactions, supporting a mechanism in which the zero-order phase results from product inhibition.

Chemical Rescue of Enzymes: Proton Transfer in Mutants of Human Carbonic Anhydrase II

In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II yields a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild-type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site-specific mutations intended to disrupt 4MI binding have demonstrated these sites to be nonproductive. In the present work, MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway.

Inhibition of Carbonic Anhydrase II by Thioxolone: A Mechanistic and Structural Study.

This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791 . Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect.

Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties

Recently, a convincing body of evidence has accumulated suggesting that the overexpression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently, there is no available X-ray crystal structure of CA IX, and this lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using (18)O-exchange and structurally using X-ray crystallography, alone and in complex with five CA sulfonamide-based inhibitors (acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide), and compared to CA II. This structural information has been evaluated by both inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX, inducing an active-site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme-specific CA IX inhibitors, which may lead to development of new therapeutic treatments of some cancers.

A T7 expression vector optimized for site-directed mutagenesis using oligodeoxyribonucleotide cassettes

Site-directed mutagenesis is widely used to examine structure/function relationships in proteins. We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps. The vectors, derived from the T7 expression vectors of Studier and his collaborators [Studier et al., Methods Enzymol. 185 (1990) 60-89], are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA. Both single-site mutants [using ssDNA and mutating oligodeoxyribonucleotides (oligos)] and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors. Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III. Production levels of greater than 60 mg of protein per liter of culture have been obtained.

Carbonic anhydrase in the adult mosquito midgut

SUMMARY We have previously demonstrated the involvement of carbonic anhydrase (CA) in larval mosquito midgut physiology. In this study, we used Hanssons histochemistry to examine the distribution of the enzyme in the midgut of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex nigripalpus, Ochlerotatus taeniorhynchus, Anopheles albimanus and Anopheles quadrimaculatus adult mosquitoes. Additionally, we quantitated CA content in the anterior and posterior midgut of adult males and females from these species using the 18O isotope exchange method coupled to mass spectrometry. We also tested the effect of CA inhibitors such as methazolamide and acetazolamide in the alkalization of the midgut in females from these species. Our results indicate that CA is present in the midgut of adults from the species studied and that it appears to be preferentially associated with the posterior midgut in both males and females. CA inhibitors appear to have a profound effect on midgut pH indicating that this enzyme might play a key role in the maintenance of this pH.

Expression and activity of carbonic anhydrase IX is associated with metabolic dysfunction in MDA-MB-231 breast cancer cells.

The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a “triple-negative,” basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydrase XII (CAXII). CAIX expression in the basal B cells is both density- and hypoxia-dependent and is correlated with carbonic anhydrase activity. Evidence is provided that CAIX contributes to extracellular acidification through studies on pH, lactic acid production, and CAIX inhibition. Together, these studies suggest that CAIX expression and activity is associated with metabolic dysfunction in MDA-MB-231 cells.

Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II.

Variants of human carbonic anhydrase II (HCA II) with amino acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and (18)O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for transfer of a proton from His64 to the zinc-bound hydroxide during catalysis were 4 and 9 μs(-1) for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8 μs(-1) for wild-type HCA II. These higher values observed for Y7F and Y7F/N67Q HCA II could not be explained by differences in the values of the pK(a) of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with a reduced level of branching in the networks of hydrogen-bonded water molecules between proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5-1.6 Å resolution. Moreover, Y7F/N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and N(ε) of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.