Biomedical subjects

Chingkuang Tu

Publications and source records attributed to Chingkuang Tu.

At least 19 recordsLinked to original sources

PubMed2006

Location of binding sites in small molecule rescue of human carbonic anhydrase II.

Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp-64 approximately 8 A from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w(r) and w(p) rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.

Binding Sites↗

PubMed2006

Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1).

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.

Aedes↗

PubMed2006

Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase.

The structures of beta class carbonic anhydrases (beta-CAs) determined so far fall into two distinct subclasses based on the observed coordination of the catalytic zinc (Zn2+) ion. The subclass of beta-CAs that coordinate Zn2+ tetrahedrally with four protein-derived ligands is represented by the structures of orthologues from Porphyridium purpureum, Escherichia coli, and Mycobacterium tuberculosis. Here we present the structure of an additional member of that subclass, that from Haemophilus influenzae, as well as detailed kinetic analysis, revealing the correspondence between structural classification and kinetic profile for this subclass. In addition, we identify a unique, noncatalytic binding mode for the substrate bicarbonate that occurs in both the H. influenzae and E. coli enzymes. The kinetic and structural analysis indicates that binding of bicarbonate in this site of the enzyme may modulate its activity by influencing a pH-dependent, cooperative transition between active and inactive forms. We hypothesize that the two structural subclasses of beta-CAs may provide models for the proposed active and inactive forms of the H. influenzae and E. coli enzymes.

Bicarbonates↗

PubMed2006

Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group.

Human salivary alpha-amylase (HSA) is a major secretory protein component of saliva and has important biological functions, including the initial digestion of starch. HSA acts as a monomer and mediates the hydrolysis of alpha-1,4-glucosidic linkages in oligosaccharides. To date, all published crystal structures of HSA have been crystallized as monomers in space group P2(1)2(1)2(1). Here, the serendipitous purification, crystallization and ultimate structure determination of a HSA non-crystallographic symmetry (NCS) dimer, while attempting to purify human carbonic anhydrase VI (HCA VI) from saliva using an affinity resin for alpha-class carbonic anhydrases, is presented. On further investigation, it was shown that HSA could only be copurified using the affinity resin in the presence of HCA VI which is glycosylated and not the non-glycosylated HCA II. The identification of the HSA crystals was carried out by peptide mapping and mass spectrometry. HSA was shown to have crystallized as an NCS dimer in space group C2, with unit-cell parameters a = 150.9, b = 72.3, c = 91.3 A, beta = 102.8 degrees. The NCS dimer crystal structure is reported to 3.0 A resolution, with a refined Rcryst of 0.228. The structure is compared with the previously reported P2(1)2(1)2(1) monomer structures and the crystal packing and dimer interface are discussed.

Crystallization↗

PubMed2005

Proton transfer in a Thr200His mutant of human carbonic anhydrase II.

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc-bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A-T200H HCAII), effectively relocating the proton shuttle residue 2 A closer to the zinc-bound hydroxide compared to wild type HCA II. The crystal structure of H64A-T200H HCA II at 1.8 A resolution shows the side chain of His200 directly hydrogen-bonded with the zinc-bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration-dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A-T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc-bound hydroxide, in analogy with proton transfer involving His64 in wild-type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc-bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen-bonded to the zinc-bound hydroxide, can adopt the correct distance geometry to support proton transfer

Amino Acid Substitution↗

PubMed2005

Human carbonic anhydrase III: structural and kinetic study of catalysis and proton transfer.

The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 A resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 A from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.

Carbonic Anhydrase III↗

PubMed2005

Proton transfer from exogenous donors in catalysis by human carbonic anhydrase II.

In the site-specific mutant of human carbonic anhydrase in which the proton shuttle His64 is replaced with alanine, H64A HCA II, catalysis can be activated in a saturable manner by the proton donor 4-methylimidazole (4-MI). From 1H NMR relaxivities, we found 4-MI bound as a second-shell ligand of the tetrahedrally coordinated cobalt in Co(II)-substituted H64A HCA II, with 4-MI located about 4.5 A from the metal. Binding constants of 4-MI to H64A HCA II were estimated from: (1) NMR relaxation of the protons of 4-MI by Co(II)-H64A HCA II, (2) the visible absorption spectrum of Co(II)-H64A HCA II in the presence of 4-MI, (3) the inhibition by 4-MI of the catalytic hydration of CO2, and (4) from the catalyzed exchange of 18O between CO2 and water. These experiments along with previously reported crystallographic and catalytic data help identify a range of distances at which proton transfer is efficient in carbonic anhydrase II.

Amino Acid Substitution↗

PubMed2005

Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II.

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer.

Alanine↗

PubMed2005

Exchange of (18)O in the reaction of peroxynitrite with CO(2).

We have observed an exchange of (18)O in the reactions of CO(2) with peroxynitrite using membrane-inlet mass spectrometry and HPLC negative electrospray ionization mass spectrometry. The exchange appeared on addition of peroxynitrite to a solution containing (18)O-labeled CO(2) in equilibrium with bicarbonate. It was observed as a temporarily enhanced rate of depletion of (18)O from CO(2), a rate that was greater than the rate of (18)O depletion caused by the hydration/dehydration cycle of CO(2). In addition, we detected the appearance of mass peaks attributed to (18)O in product NO(3)(-). As a further measure of the (18)O exchange, there was a redistribution of (18)O such that the ratio of doubly to singly labeled CO(2) could not be described by the binomial expansion. This is not due to the hydration/dehydration cycle of CO(2) but most likely to recycling of CO(2) in the reaction with peroxynitrite. This (18)O exchange associated with the reactions of CO(2) and peroxynitrite may open a new methodology for studying this significant process.

Carbon Dioxide↗

PubMed2005

Carbonic anhydrase in the adult mosquito midgut.

We have previously demonstrated the involvement of carbonic anhydrase (CA) in larval mosquito midgut physiology. In this study, we used Hansson's 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.

Acetazolamide↗

PubMed2004

Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana.

We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconversion in alpha-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A. thaliana beta-carbonic anhydrase provide direct evidence for the role of this residue in beta-carbonic anhydrase. Namely, the mutation of Gln-158 to Ala results in a significant decrease in the maximal k(cat) (33% of wild type) at steady state and the maximal rate of CO(2)(-) HCO(2)(-) exchange at chemical equilibrium as measured by R(1)/[E] (7% of wild type), while leaving the maximal rate of H(+) transfer, as measured by k(cat) at steady state, or R(H(2)O)) at chemical equilibrium, largely unaffected.

Arabidopsis↗

PubMed2004

Activation of carbonic anhydrase II by active-site incorporation of histidine analogs.

The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.

Binding Sites↗

PubMed2004

Alkalization of larval mosquito midgut and the role of carbonic anhydrase in different species of mosquitoes.

We have previously demonstrated the involvement of carbonic anhydrase (CA) in the alkalization mechanism of the Aedes aegypti larval midgut. In this study, we used Hansson's histochemistry to examine the distribution of the enzyme in the midgut of six different species of mosquito larvae (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex nigripalpus, Ochlerotatus taeniorhynchus, Anopheles quadrimaculatus). Additionally, we quantitated CA content in the gastric caeca, anterior and posterior midgut of fourth instar larvae 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 for these species. Our results indicate that CA is present in the larval midgut of the species studied and that it appears to be associated with the posterior midgut and gastric caeca in some species and with the anterior midgut in others. CA inhibitors appear to have a profound effect on the alkalization mechanism of the midgut with lethal consequences for most of the species tested.

Acetazolamide↗

PubMed2002

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class.

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.

Alanine↗

PubMed2002

The refined atomic structure of carbonic anhydrase II at 1.05 A resolution: implications of chemical rescue of proton transfer.

Using synchrotron radiation and a CCD detector, X-ray data have been collected at 100 K for the His64Ala mutant of human carbonic anhydrase II complexed with 4-methylimidazole (4-MI) to a maximal 1.05 A resolution, allowing full anisotropic least-squares refinement. The refined model has a conventional R factor of 15.7% for all reflections. The C(alpha) coordinates of the model presented here have an r.m.s. deviation of 0.10 A relative to the previously determined structure at 1.6 A resolution. Several amino-acid residues (six of the 255 observed) have been identified with multiple rotamer side-chain conformations. C, N and O atoms can be differentiated with selective electron-density map contouring. The estimated standard deviations for all main-chain non-H atom bond lengths and angles are 0.013 and 0.030 A, respectively, based on unrestrained full-matrix least-squares refinement. This structure gives detailed information about the tetrahedrally arranged zinc ion coordinated by three histidine N atoms (His94 N(epsilon 2), His96 N(epsilon2) and His119 N(delta1)) and a water/hydroxide, the multiple binding sites of the proton chemical rescue molecule 4-MI and the solvent networks linking the zinc-bound water/hydroxide and 4-MI molecules. This structure presents the highest resolution structure of a carbonic anhydrase isozyme so far determined and adds to the understanding of proton-transfer processes.

Amino Acid Substitution↗

PubMed2002

Proton transfer within the active-site cavity of carbonic anhydrase III.

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.

Binding Sites↗

PubMed2002

Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana.

We have cloned and overexpressed a truncated, recombinant form of beta-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by (18)O isotope exchange methods. The wild-type enzyme has a maximal k(cat) for CO2 hydration of 320 ms(-1) and is rate limited by proton transfer involving two residues with apparent pK(a) values of 6.0 and 8.7. The mutant enzyme H216N has a maximal k(cat) at high pH that is 43% that of wild type, but is only 5% that of wild type at pH 7.0. (18)O exchange studies reveal that the effect of the mutations H216N or Y212F is primarily on proton transfer steps in the catalytic mechanism and not in the rate of CO2-HCO3- exchange. These results suggest that residues His-216 and Tyr-212 are both important for efficient proton transfer in A. thaliana carbonic anhydrase.

Amino Acid Substitution↗

PubMed2002

Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase.

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.

Binding Sites↗