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Biomedical subjects

S Xiang

Publications and source records attributed to S Xiang.

At least 19 recordsLinked to original sources
PubMed2001

On the relationship between protein stability and folding kinetics: a comparative study of the N-terminal domains of RNase HI, E. coli and Bacillus stearothermophilus L9.

There is currently a great deal of interest in proteins that fold in a single highly cooperative step. Particular attention has been focused on elucidating the factors that govern folding rates of simple proteins. Recently, the topology of the native state has been proposed to be the most important determinant of their folding rates. Here we report a comparative study of the folding of three topologically equivalent proteins that adapt a particularly simple alpha/beta fold. The folding kinetics of the N-terminal domain of RNase HI and the N-terminal domain of the ribosomal protein L9 from Escherichia coli (eNTL9) were compared to the previously characterized N-terminal domain of L9 from Bacillus stearothermophilus (bNTL9). This 6.2 kDa protein, which is one of simplest examples of the ABCalphaD motif, folds via a two-state mechanism on the millisecond to submillisecond time scale. The RNase HI domain and bNTL9 have very similar tertiary structures but there is little similarity in primary sequence. bNTL9 and eNTL9 share the same biological function and a similar primary sequence but differ significantly in stability. Fluorescence-detected stopped-flow experiments showed that the three proteins fold in a two-state fashion. The folding rates in the absence of denaturant were found to be very different, ranging form 21 s(-1) to 790 s(-1) at 10 degrees C. The diverse folding rates appear to reflect large differences in the stability of the proteins. When compared at an isostability point, the folding rates converged to a similar value and there is a strong linear correlation between the log of the folding rate and stability for this set of proteins. These observations are consistent with the idea that stability can play an important role in dictating relative folding rates among topologically equivalent proteins.

Amino Acid Motifs↗
PubMed2001

The distinct roles of T cell-derived cytokines and a novel follicular dendritic cell-signaling molecule 8D6 in germinal center-B cell differentiation.

Germinal center-B (GC-B) cells differentiate into memory B cells and plasma cells (PC) through interaction with T cells and follicular dendritic cells (FDC). Activated T cell and FDC play distinct roles in this process. The detailed kinetic experiments revealed that cytokines secreted by activated T cells determined the pathway of GC-B cell differentiation. IL-4 directs GC-B cells to differentiate into memory B cells, whereas IL-10 steers them into PC. FDC/HK cells do not direct either pathway, but provide signals for proliferation of GC-B cells. A novel FDC-signaling molecule 8D6 (FDC-SM-8D6) produced by FDC augments PC generation in the GC. FDC-SM-8D6-specific mAb blocked PC generation and IgG secretion but not memory B cell proliferation. COS cells expressing FDC-SM-8D6 enhanced GC-B cell proliferation and Ab secretion, which was blocked by mAb 8D6. In the cultures with B cell subsets, PC generation was inhibited by mAb 8D6 in the cultures with CD27(+) B cells but not in the culture with CD27(-) B cells, suggesting that CD27(+) PC precursor is the specific target of FDC-SM-8D6 stimulation.

Antibodies, Blocking↗
PubMed2001

Binding of low density lipoproteins to lipoprotein lipase is dependent on lipids but not on apolipoprotein B.

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.

Animals↗
PubMed2001

The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin.

BACKGROUND: Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in archaea, eubacteria, and eukaryotes. In humans, genetic abnormalities in the biosynthetic pathway result in Moco deficiency, which is accompanied by severe neurological symptoms and death shortly after birth. The Escherichia coli MoeA and MogA proteins are involved in the final step of Moco biosynthesis: the incorporation of molybdenum into molybdopterin (MPT), the organic pyranopterin moiety of Moco. RESULTS: The crystal structure of E. coli MoeA has been refined at 2 A resolution and reveals that the highly elongated MoeA monomer consists of four clearly separated domains, one of which is structurally related to MogA, indicating a divergent evolutionary relationship between both proteins. The active form of MoeA is a dimer, and a putative active site appears to be localized to a cleft formed between domain II of the first monomer and domains III and IV of the second monomer. CONCLUSIONS: In eukaryotes, MogA and MoeA are fused into a single polypeptide chain. The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains. This multidomain arrangement could provide a structural basis for its functional diversity. The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.

Amino Acid Sequence↗
PubMed2000

Effects of hepatic artery chemotherapeutic embolization combined with perfusing LAK cells into hepatic artery after radical operation of liver cancer.

OBJECTIVE: To evaluate the effects of hepatic artery chemotherapeutic embolization combined with perfusing LAK cells/interleukin 2 into hepatic artery after radical operation of liver cancer. METHODS: Random divide 42 cases into two groups who had accepted radical operation of liver cancer: 21 cases who accepted hepatic artery chemotherapeutic embolization combined with perfusing LAK cells/interleukin 2 into hepatic artery as observing group, 21 cases accepted simply hepatic artery chemotherapeutic embolization as control group. RESULTS: The intrahepatic recurrence rates of 1, 2, and 3 years were 0, 19.05%, 57.14% in observing group, lower than 28.57%, 47.62%, 85.71% of control group (chi(2)=4.86, 3. 86 and 4.20 respectively, P<0.05). The survival rates of 2 and 3 years were 85.71%, 61.90% in observing group, higher than 57.14%, 28. 57% of control group (chi(2)=4.20 and 4.71, P<0.05). CONCLUSION: Hepatic artery chemotherapeutic embolization combined with perfusing LAK cells/interleukin 2 into hepatic artery is an effective therapeutic method to control intrahepatic recurrence rates and raise the survival rates of the patients.

Adult↗
PubMed1998

Identification of a novel transcriptional silencer in the protein-coding region of the human CYP2C9 gene.

A novel regulatory element (27 bp) which confers transcriptional repression was identified within the protein-coding region immediately after the translation start codon in the human cytochrome P450 (CYP) 2C9 gene. Deletion of this element increased transcriptional activity in HepG2 cells by transient transfection assay. Nuclear protein extracts from HepG2 cells and human liver were found in electrophoretic mobility shift assays to bind specifically to the 27 bp element. A putative binding protein was partially purified by DNA-affinity chromatography and was determined by Southwestern blotting to have a molecular weight of approx. 100 kDa. Studies with mutated competitor oligonucleotides established that binding of the nuclear protein to the 27 bp cis-element was dependent upon two 6 bp direct repeats (5'-CTTGTG-3') that were separated by three bases. It is possible that this novel cis-acting element may be involved in the negative regulation of CYP2C9.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Th1/Th2 cytokine profiles in the nasopharyngeal lymphoid tissues of children with recurrent otitis media.

The cytokine profile in adenoidal lymphoid tissue was studied in 22 patients. Lymphocytes from adenoid tissues and peripheral blood were submitted for cytokine assays using an enzyme-linked immunosorbent assay kit for interferon-gamma, interleukin (IL)-2, IL-4, IL-5, IL-6, and IL-10. Adenoidal lymphocytes appear to produce significantly less Th1 cytokines (IL-2, interferon gamma) compared to the patient's peripheral blood lymphocytes, whereas IL-4 and IL-5 (Th2 cytokines) appear to be synthesized to the same extent as, if not slightly more than, in the homologous peripheral blood lymphocytes. Because the relationship between Th1 and Th2 cytokines is extremely important in modulating the immune response, it is advisable to determine the role of the cytokine profiles of T-lymphocytes in the nasopharynx and its relationship to the development of inflammation of the eustachian tube and middle ear.

Adenoids↗
PubMed1997

The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization.

Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to bind the incoming 4-NH2 group represents the site for the leaving ammonia molecule. The conserved Pro 128 accommodates both moieties by orienting the carbonyl group of the previous residue. (2) The Glu 104 carboxylate group rotates from its hydrogen bond to the O4 hydroxyl group in transition-state analog complexes, forming a new hydrogen bond to the leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to the leaving amino group, promoting enol-to-keto isomerization of the product. (3) Difference Fourier comparisons with transition-state complexes indicate that the pyrimidine ring rotates toward the zinc by approximately 10 degrees. The active site thus "pulls" the ring and 4-NH2 group in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the N1-C1' glycosidic bond bends by approximately 19 degrees out of the ring plane. This distortion may "spring-load" the product complex and promote dissociation. Failure to recognize a similar distortion could explain an earlier crystallographic interpretation of the adenosine deaminase-inosine complex [Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-Sgamma132 bond, which lengthens in transition-state complexes, shortens as the O4 atom returns to a state of lower negative charge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of negative charge on the oxygen nucleophile during catalysis.

Binding Sites↗
PubMed1997

[Application of serum bile acid chromatography to the diagnoses of liver diseases].

In order to explore the specificity of serum bile acid (SBA) chromatograph in the diagnoses of different kinds of hepatosis, we investigated by means of gas chromatography the changes of serum bile acids in workers who exposed to hexogen or chloroethylene and in patients who suffered from hepatosis such as acute jaundice hepatitis, chronic active hepatitis, cirrhosis and liver cancer. The results revealed different disturbances of SBA occurring in the liver injuries induced by the two kinds of hepatotoxicant. Serum lithocholic acid (LCA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) in workers exposed to hexogen and wre significantly different from those of the control group respectively (P < 0.01, P < 0.01, P < 0.05); on the other hand, only serum LCA and DCA went up in workers exposed to chloroethylene (P < 0.0005, P < 0.001). The main changes both concentrated on the secondary bile acids. In acute jaundice hepatitis, chronic active hepatitis, cirrhosis and liver cancer, serum LCA, DCA, CDCA and cholic acids (CA) all went up in different degrees compared with the control group respectively (P < 0.005, P < 0.025, P < 0.005, P < 0.005). But no difference was noted among the 4 kinds of bile acids (P > 0.5), except that between CA and CDCA. These provided the evidence of the diagnosis and identification of clinical hepato-biliary diseases and occupational liver injures.

Cholic Acids↗
PubMed1996

Cytidine deaminase complexed to 3-deazacytidine: a "valence buffer" in zinc enzyme catalysis.

The cytidine deaminase substrate analog inhibitor 3-deazacytidine binds with its 4-amino group inserted into a site previously identified as a probable binding site for the leaving ammonia group. Binding to this site shifts the pyrimidine ring significantly further from the activated water molecule than the position it occupies in either of two complexes with compounds capable of hydrogen bonding at the 3-position of the ring [Xiang et al. (1995) Biochemistry 34, 4516-4523]. Difference Fourier maps between the deazacytidine, dihydrozebularine, and zebularine--hydrate inhibitor complexes suggest that the ring itself moves successively toward the activated water, leaving the amino group behind in this site as the substrate complex approaches the transition state. They also reveal systematic changes in a single zinc-sulfur bond distance. These correlate with chemical changes expected as the substrate approaches the tetrahedral transition state, in which the zinc-activated hydroxyl group develops maximal negative charge and forms a short hydrogen bond to the neighboring carboxylate group of Glu 104. Empirical bond valence relationships suggest that the Zn-S gamma 132 bond functions throughout the reaction as a "valence buffer" that accommodates changing negative charge on the hydroxyl group. Similar structural features in alcohol dehydrogenase suggest that analogous mechanisms may be a general feature of catalysis by zinc enzymes.

Catalysis↗
PubMed1996

Representing stereochemical information in macromolecular electron-density distributions by multi-dimensional histograms.

Previous studies have demonstrated the value of ideal electron-density histograms as targets for the corresponding histograms of experimental electron-density maps. The electron-density histogram makes use of density values as independent objects, and no relationship between them is taken into account. Extension to include the relationships between neighboring density values leads naturally to a multi-dimensional histogram defined as the joint frequency of the density values and their higher order derivatives. We show here that the multi-dimensional histogram including additional dimensions composed of the gradient magnitude and Laplacian of the density is minimally dependent on molecular folding and packing, and captures substantially more stereochemical information than the conventional electron-density histogram. The gradient histogram appears to be much more sensitive to phase errors than the conventional electron-density histogram. Potential uses of the multi-dimensional histogram include improved targets for density modification and more reliable figures of merit for evaluating correct phases.

Journal Article↗
PubMed1996

Modulation of B-cell immunoglobulin synthesis by retinoic acid.

Retinoic acid (RA) and its parent compound retinol (ROH, vitamin A) have been recognized as important immunopotentiating agents since the early 1900s. We have focused our studies on the effects of retinoids on B-cell immune function in the newborn infant. The response of cord blood mononuclear cells (CBMC) to formalinized Cowan I strain Staphylococcus aureus (SAC), a T-cell-dependent factor for inducing the differentiation of B cells into immunoglobulin (Ig)-secreting cells, was used as a model system for studying whether RA could alter the immunoglobulin synthesis of newborn B lymphocytes. The addition of RA to SAC-stimulated CBMC cultures produced a 2- to 47-fold increase in IgM synthesis. An ELISA-spot assay showed that the RA-induced enhancement in Ig synthesis was due to the recruitment of more B cells to differentiate into Ig-secreting cells. Whereas RA enhanced IgM production of CBMC stimulated with SAC, RA augmented only IgG production of SAC-stimulated adult peripheral blood mononuclear cells (PBMC). To determine if the differences in dose-response characteristics between CBMC and adult PBMC resided within the target cell, i.e., the B cell, T-cell-enriched and T-cell-depleted (B-cell) fractions from CBMC and adult PBMC were cocultured in various combinations. The isotype, i.e., IgM vs IgG, and the dose-response curve characteristics were intrinsic to the responding B-cell source, i.e., newborn vs adult. Highly purified T cells from CBMC, when preincubated for 36 hr with RA, enhanced IgM synthesis of cord blood B cells. Supernatants from purified T cells generated a factor which could enhance B-cell synthesis. Although interleukin (IL)-2, IL-4, and IL-6 could not be detected by ELISA in the T-cell-derived supernatants, RA probably generates a cytokine/interleukin from T cells which modulates B-cell Ig secretion. RA can also act directly on B cells as evidenced by the augmentation in Ig synthesis of Epstein-Barr virus (EBV)-transformed B-cell lines. These data suggest that RA can have a direct effect on B cells. Since increased proliferation (numbers) of lymphoblastoid B cells was not responsible for the increased amounts of Ig in the supernatant fluids, we examined whether cytokines secreted by EBV-transformed B cells could be acting as an autocrine factor in increasing Ig synthesis. EBV-transformed B-cell clones incubated with RA for 6 days produced a 20- to 45-fold increase in IL-6. An understanding of the mechanisms by which RA enhances B-cell immune function may lead to the use of RA or its derivatives in patients with immune deficiencies and in preterm infants with immature immune systems.

Adjuvants, Immunologic↗
PubMed1996

The effects of retinoic acid on immunoglobulin synthesis: role of interleukin 6.

Retinoic acid (RA) and its parent compound, retinol (ROH, vitamin A), have been recognized as important immunopotentiating agents. Previous studies from our laboratory have demonstrated that RA can augment formalin-treated Staphylococcus aureus (SAC)-stimulated immunoglobulin (Ig) synthesis of cord blood mononuclear cells (CBMC). To determine the mechanism(s) by which RA modulates Ig synthesis, we studied the effects of RA on B cells and cytokine production. The addition of RA (10(-5) to 10(-10) M) to Epstein-Barr virus (EBV)-transformed B-cell clones derived from either adult or cord blood B cells augmented Ig secretion twofold. In contrast, cell proliferation was inhibited as measured by 3H-thymidine incorporation. We evaluated two cytokines known to be constitutively produced by EBV cell lines, IL-1 and IL-6. While RA had no effect on IL-1 production, IL-6 synthesis was greatly enhanced (20- to 45-fold), which was also reflected by an increase in steady-state mRNA levels for IL-6 but not TNF-alpha or TGF-beta on Northern blot analysis. Polyclonal rabbit anti-IL-6 antibodies were used to block the augmenting effects of RA on Ig synthesis of adenoidal B cells. RA-induced augmentation in IgG and IgA synthesis was blocked 58 and 29%, respectively, by anti-IL-6 antibodies. These studies suggest that the enhancing effects of RA on Ig synthesis are mediated, at least in part, by the autocrine or paracrine effects of IL-6 on B-cell differentiation.

Adult↗
PubMed1996

Interrelationship between magnesium and potassium in preventing myocardial ischemia-reperfusion arrhythmia.

OBJECTIVE: To assess the interrelationship between Mg2+ and K+ in preventing reperfusion arrhythmia (RA). MATERIAL AND METHODS: 72 rat hearts were randomly divided into 6 equal groups, and perfused with various combinations of Mg2+ and K+ in the perfusate by Langendorff method. Each heart was subjected to coronary occlusion for 15 minutes followed by 12-minute reperfusion. The incidence of RA was recorded. The concentration of K+ and malondialdehyde (MDA) in the coronary effluent before and after reperfusion were measured with increasing concentrations of Mg2+ and fixed concentration of K+. RESULTS: All forms of ventricular arrhythmias occurred after reperfusion but with decreased incidence by increasing Mg2+ and K+. When both Mg2+ and K+ were in high level, no VF appeared on reperfusion. However, when they were both in low level, 100% of hearts developed severe arrhythmia. The concentration of K+ and MDA in the coronary effluent was markedly increased compared with that before reperfusion, and the loss of K+ and malondialdehyde (MDA) decreased with increasing Mg2+. CONCLUSIONS: Mg2+ may prevent or reduce the incidence of RA by maintaining the integrity of intracellular K+ and by reducing the lipid peroxide reaction.

Animals↗
PubMed1995

Transition-state selectivity for a single hydroxyl group during catalysis by cytidine deaminase.

Cytidine deaminase binds transition-state analog inhibitors approximately 10(7) times more tightly than corresponding 3,4-dihydro analogs containing a proton in place of the 4-hydroxyl group. X-ray crystal structures of complexes with the two matched inhibitors differ only near a "trapped" water molecule in the complex with the 3,4-dihydro analog, where contacts are substantially less favorable than those with the hydroxyl group of the transition-state analog. The hydrogen bond between the hydroxyl group and the Glu 104 carboxylate shortens in that complex, and may become a "low-barrier" hydrogen bond, since at the same time the bond between zinc and the Cys 132 thiolate ligand lengthens. These differences must therefore account for most of the differential binding affinity related to catalysis. Moreover, the trapped water molecule retains some of the binding energy stabilizing the hydroxyl group in the transition-state analog complex. To this extent, the ratio of binding affinities for the two compounds is smaller than the true contribution of the hydroxyl group, a conclusion with significant bearing on interpreting difference free energies derived from substituent effects arising from chemical modification and/or mutagenesis.

Catalysis↗
PubMed1994

Overcoming non-isomorphism by phase permutation and likelihood scoring: solution of the TrpRS crystal structure.

Entropy maximization to maximum likelihood, constrained jointly by the best available experimental phases and by a sufficiently good envelope, can bring about substantial model-independent map improvement, even at medium (3.1 A) resolution [Xiang, Carter, Bricogne & Gilmore (1993). Acta Cryst. D49, 193-212]. In the crystal structure determination of the Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS), however, the following had to be dealt with simultaneously: (1) a serious lack of isomorphism in the heavy-atom derivatives, resulting in large starting-phase errors; and (2) an initially poorly known molecular envelope. Because the constraints--both phases and envelope--were insufficiently well determined at the outset, maximum-entropy solvent flattening as previously applied was unsuccessful. Rather than improving the maps, it led to a deterioration of their quality, accompanied by a dramatic decrease of the log-likelihood gain as phases were extended from about 5 A resolution to the 2.9 A limit of the diffraction data. This deadlock was broken by the identification of strong reflections, which were initially unphased and which were inaccessible by maximum-entropy extrapolation from the phased ones, and by permutation of the phases of these reflections so as to sample the space of possible electron-density and envelope modifications they represented. Permutation was carried out by successive full and incomplete factorial designs [Carter & Carter (1979). J. Biol. Chem. 254, 12219-12223] for 28 strong reflections selected in decreasing order of their 'renormalized' structure-factor amplitudes. The permuted reflections included one reflection for which the probability distribution from multiple isomorphous replacement with anomalous scattering (MIRAS) indicated an incorrect phase with a high figure of merit and which consequently had a large renormalized structure factor. A similar permutation was carried out for six different binary choices related to the calculation and description of the molecular envelope. Permutation experiments were scored using the log-likelihood gain and contrasts for each main effect were analyzed by multiple-regression least squares. Student t tests provided significant and reliable indications for a large majority of the permuted reflections and for all six hypotheses related to the molecular envelope. The resulting phase improvement made it possible to assign positions (hitherto unobtainable) for nine of the ten selenium atoms in an isomorphous difference Fourier map for selenomethionine-substituted TrpRS crystals and hence to solve the structure. Phase-permutation methods continued to be useful in producing improved maps from all the available isomorphous-replacement phase information and therefore played a critical role in solving the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Bayes Theorem↗
PubMed1994

Cytidine deaminase. The 2.3 A crystal structure of an enzyme: transition-state analog complex.

We have solved the structure of Escherichia coli cytidine deaminase (CDA) complexed to the transition state analog, 5-fluoroprimidin-2-one riboside. The monomer of the alpha 2 CDA dimer is composed of a small N-terminal alpha-helical domain with no obvious connection to the active sites, and two, larger, core domains. The two core domains have nearly identical tertiary structures and are related by approximate 2-fold symmetry, but lack internal amino acid sequence homology. Comparison of the core domain structure with known structures by sequence homology and structural compatibility searches suggests that the CDA tertiary structure cannot be superimposed on any known protein structure. The two active sites per dimer are formed across the subunit interface. The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket and the structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent. The deeply buried zinc-binding site is formed by a novel "topological switch point" at the amino termini of two alpha-helices in consecutive alpha-beta-alpha-beta segments. The transition state analog is bound as a covalent hydrate at C4. The inhibitor hydroxyl oxygen atom interacts both with the zinc atom and the Glu104 carboxylate group, affording high differential affinity for the hydroxyl group relative to a hydrogen atom, in a manner reminiscent of that observed in adenosine deaminase (ADA). Unlike the latter enzyme, the zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group. Moreover, the inhibitor stereochemistry is of the opposite hand from that of the corresponding ADA inhibitor at C4(R), but is the same at the hydroxyl group O4(S). A consequence of these stereochemical differences is that in CDA a single conserved carboxylate side-chain, Glu104, can provide all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group. The differences in zinc ligands, ligand-binding stereochemistry, and tertiary structures of CDA and ADA strongly suggest that the common features of transition state stabilization arose by convergent evolution.

Amino Acid Sequence↗