Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).     

The 1.8 A structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism

Intramolecular trans-sialidase from leech (Macrobdella decora) is the first member of the sialidase superfamily found to exhibit strict specificity towards the cleavage of terminal Neu5Acalpha2-->3Gal linkage in sialoglycoconjugates. Its release of 2,7-anhydro-Neu5Ac instead of Neu5Ac indicates that it catalyzes an intramolecular trans-sialosyl reaction. Crystal structures of its complexes with an inactive substrate analogue 2-propenyl-Neu5Ac, and with the product 2,7-anhydro-Neu5Ac, have been determined to 1.8 A resolution. The boat conformation of the pyranose observed in the complexes supports the proposed enzymatic mechanism that O7 of an axial 6-glycerol group attacks the positively charged C2 of the intermediate. A generalized mechanism is proposed for the sialidase superfamily.     

Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase

A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.     

Modeling the interaction between FK506 and FKBP12: a mechanism for formation of the calcineurin inhibitory complex

FK506 is a naturally occurring immunosuppressant whose mode of action involves formation of an initial complex with the cytosolic protein FKBP12. The composite surface of this complex then binds to and inhibits the protein phosphatase calcineurin (PP2B). To investigate why FK506 does not inhibit calcineurin directly we have conducted molecular modeling and conformational studies on published structures of FK506 both alone and in complex with FKBP12. From studies of the structure of FK506 in CDCl3 and Z-Arg32-ascomycin in water (a water soluble analogue of FK506) we suggest that the FK506 molecule can be viewed as consisting of three separate regions. The pipecolate region which extends from C24 to C10 including the pipecolate ring shows strongly conserved conformation in both solvents. The loop region which extends from C25 to C16 shows general conservation of the loop structure and the pyranose region made up of the pyranose ring and C15-C17 which shows highly variable conformation depending on solvent. Comparison of the structure of Z-Arg32-ascomycin in water with structures of FK506 bound to FKBP12 indicate that the conformation of the pipecolate region is conserved during the binding process. The conformation of the loop region was generally conserved but a significant reduction (approximately 1.7 A) in the diameter of the loop in the bound structure was observed. The conformation of the pyranose ring and C15-C17 region was found to be significantly altered in the bound structure resulting in displacements of the C13 and C15 methoxyl groups of 2.8 and 3.5 A, respectively. From computer models and molecular dynamics simulations of interactions between FK506 and FKBP12 we suggest that the conformational changes observed in bound FK506 are induced by the interaction between the 80's loop of FKBP12 and the pyranose ring of FKBP12. These interactions result in the formation of a complex with the both correct shape and surface polarity for interaction with calcineurin.     

Molecular modelling studies of lysozyme catalysed hydrolysis of synthetic substrates

Kinetic data for the lysozyme catalysed hydrolysis of aryl chitooligosides were surveyed. Both electron-donating and electron-withdrawing substituents on the departing aryl aglycones enhance the rate of hydrolyses. The parallel pH-rate profiles implicate that identical catalytic residues are involved in the hydrolytic fission of the glycosyl-aryloxy bond of these two groups of synthetic substrates. Molecular modelling studies of lysozyme complexes with aryl diN-acetyl chitobiosides and their intermediates were performed. The two synthetic substrates bearing aryl aglycones with opposite electronic effects bind to the active site of lysozyme in different conformations. Based on the energetic and geometric considerations, the oxocarbonium ion whose pyranose ring D in a sofa conformation is the most plausible reaction intermediate for the lysozyme catalysed hydrolysis of the synthetic substrates. The modelling study also suggests that considerable conformational changes of both the lysozyme binding site and the chitobiosyl group accompany the formation of the glycosyl enzyme intermediate. In particular, the chitobiosyl group undergoes a dislocation of the pyranose ring C from the subsite C and a constraint of the pyranose ring D to form a boat conformer.     

Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus

We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.     

Substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus

The substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus was investigated using, 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Evaluation of the affinities of ten imaginary subsites by the method of Suganuma et al. [1978, J. Biochem. (Tokyo) 84, 293--316] pointed out that the substrate-binding site of the enzyme is composed of four subsites and that the catalytic groups are localized in the centre. The imaginary subsites on the left-hand side of the binding site ('non-reducing-end' side) showed little or no affinity to bind xylosyl residues. For the subsites on the right-hand side of the binding site ('reducing-end' side) negative values of affinity were obtained, which means this region of the enzyme is unfavourable for complexing with xylosyl residues. As a consequence of the asymmetric distribution of negative values of affinity around the binding site, the enzyme displays a strong preference for attacking near the reducing end of the substrate. Regardless of the length of [1-3H]xylooligosaccharides, [1-3H]xylobiose was the prevailing reaction product at an early stage of hydrolysis, and frequency distribution of bond cleavage decreased from the second glycosidic bond towards the non-reducing end. Additional information on the substrate-binding site of C. albidus beta-xylanase was obtained by evaluating the efficiency of xylose, xylobiose, methyl beta-D-xyloside and phenyl beta-D-xyloside to serve as glycosyl acceptors in the transglycosylic reactions proceeding at high concentrations of xylotriose.     

Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue

The 2.4-A resolution crystal structure of a dominantly active form of the small guanosine triphosphatase (GTPase) RhoA, RhoAV14, complexed with the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), reveals a fold similar to RhoA-GDP, which has been recently reported (Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703), but shows large conformational differences localized in switch I and switch II. These changes produce hydrophobic patches on the molecular surface of switch I, which has been suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of the antiparallel beta-sheet between switches I and II. Key residues that produce these conformational differences were identified. In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of the Rho family, including Rac1, but with several variations in details. These sites also display large displacements from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgammaS by the GTPase contains water-mediated hydrogen bonds, which seem to be common in the Rho family. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as guanine nucleotide exchange factor (GEF) and guanine nucleotide dissociation inhibitor (GDI).     

An improved method for preparing lysozyme with chemically 13C-enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis

Jones et al. have reported that the epsilon-carbons of methionine residues in myoglobin can be enriched with stable isotope (13C) in two steps, i.e., methylation of methionine residues with 13CH3I in the protein and thiolysis using dithiothreitol [Jones, W.C., Rothgeb, T.M., and Gurd, F.R.N. (1976) J. Biol. Chem. 251,7452-7460]. Using their method, we failed to prepare active lysozyme in which the epsilon-carbons of methionine residues are enriched with 13C, because many side reactions took place under the thiolysis condition (pH 10.5, 37 degrees C). When we employed 2-aminothiophenol as a reagent for thiolysis, the reduction proceeded under a weakly acidic condition to afford fully active lysozyme, in which the epsilon-carbons of two methionine residues were enriched with 13C, in a 30% yield. Analysis of the 13C-edited NOESY spectra of 13C-enriched methionine lysozyme in the absence and presence of a substrate analogue indicated the occurrence of conformational change around Met 105 in lysozyme.     

The role of non-catalytic binding subsites in the endonuclease activity of bovine pancreatic ribonuclease A

Bovine pancreatic ribonuclease A catalyzes the depolymerization of RNA. There is much evidence that several subsites, in addition to the main catalytic site, are involved in the formation of the enzyme-substrate complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage shows that the enzyme does not act in a random fashion but rather prefers the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6-7 residues apart from the end of the chain to be preferentially cleaved by ribonuclease A. The results demonstrate the model of the cleavage of an RNA chain based on the cooperative binding between the multisubsite binding structure of ribonuclease A and the phosphates of the polynucleotide (Parés, X., Nogués, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol. 26, pp. 89-103, Portland Press Ltd., London). The contribution to the enzymatic process of the non-catalytic phosphate-binding subsite (p2) adjacent to the catalytic center has been analyzed in p2 chemically modified ribonuclease A or by means of site-directed mutagenesis. In both cases deletion of p2 abolishes the endonuclease activity of ribonuclease A, which is substituted by an exonuclease activity. All these results support the role of the multisubsite structure of the enzyme in the endonuclease activity and in the catalytic mechanism.     

The propeptide of the vitamin K-dependent carboxylase substrate accelerates formation of the gamma-glutamyl carbanion intermediate

Vitamin K-dependent carboxylase catalyzes the post-translational gamma-carboxylation of 9-12 glutamyl residues of several blood coagulation proteins. Carboxylase purified from Chinese hamster ovary (CHO) cells as a recombinant FLAG-carboxylase fusion protein [Sugiura, I., et al. (1996) J. Biol. Chem. 271, 17837-17844] was utilized with pentapeptide substrate FL[3H-R,S]EAL with high specific radioactivity to probe the timing of glutamyl Cgamma-3H cleavage relative to Cgamma-COO- bond formation by 14CO2 incorporation rates. Studies were conducted over a range of NaH14CO3 concentrations to assess uncoupling of gamma-glutamyl carbanion formation and over a range of concentrations of ProPT18, the 18-residue peptide corresponding to the -18 to -1 propeptide region of prothrombin known to affect the catalytic efficiency of carboxylase. At saturation, ProPT18 accelerates Cgamma-3H cleavage 11-13-fold and Cgamma-14CO2- formation 6-7-fold, converting a Cgamma-3H cleavage/Cgamma-14CO2- formation ratio of 1.2-1.4 in the absence of ProPT18 to 2.3-2.8 in its presence, a relative increase in and uncoupling of Cgamma-3H cleavage from C-C bond formation. When the HCO3- concentration was varied, the V/K3H+/V/K14CO2 ratios rose as HCO3- fractional saturation dropped to a ratio of 9.3-10.8/l at low bicarbonate, indicating an uncoupling of nine out of ten gamma-glutamyl carbanion formations from carboxylative capture, consistent with prior reports on microsomal enzyme [Larson, A. E., et al. (1981) J. Biol. Chem. 256, 11032-11035]. These results with pentapeptide substrate FLEAL validate reversible gamma-glutamyl carbanion formation by pure carboxylase and indicate the ProPT18 increase in catalytic efficiency is in selective lowering of an energy barrier preceding the gamma-glutamyl carbanion intermediate.     

The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium. Characterization of the ATPase activity associated with the purified MalK subunit

The ATPase activity associated with the purified MalK subunit of the maltose transport complex of Salmonella typhimurium, a bacterial ATP-Binding Cassette (ABC) transporter (Walter, C., H?ner zu Bentrup, K., and Schneider, E. (1992) J. Biol. Chem. 267, 8863-8869), was characterized in detail. The analysis of the kinetics of ATP hydrolysis yielded a Km value of 70 +/- 4 microM and a Vmax of 1.3 +/- 0.3 mumol/min/mg of protein. Both GTP and CTP also served as substrates. While MalK exhibited nearly the same affinity for GTP as for ATP, the Michaelis constant for CTP as a substrate was much higher. ATP hydrolysis was strongly dependent on the presence of Mg2+ ions. Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+. The ATPase activity was optimal at slightly alkaline pH and was stimulated in the presence of both glycerol (7.5%) and dimethyl sulfoxide (Me2SO) (5%). ADP and the non-cleavable substrate analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) were identified as competitive inhibitors. The MalK-ATPase was resistant to specific inhibitors of F-, P-, and V-type ATPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomycin A1. In contrast, micromolar concentrations of the sulfhydryl reagent N-ethylmaleimide strongly inhibited the enzymatic activity. This inhibition was blocked in the presence of ATP. These results suggest that the intrinsic ATPase activity of purified MalK can be clearly distinguished from other ATP-hydrolyzing enzymes, e.g. ion-translocating ATPases.     

Interaction of substrate uridyl 3',5'-adenosine with ribonuclease A: a molecular dynamics study

A wealth of information available from x-ray crystallographic structures of enzyme-ligand complexes makes it possible to study interactions at the molecular level. However, further investigation is needed when i) the binding of the natural substrate must be characterized, because ligands in the stable enzyme-ligand complexes are generally inhibitors or the analogs of substrate and transition state, and when ii) ligand binding is in part poorly characterized. We have investigated these aspects in the binding of substrate uridyl 3',5'-adenosine (UpA) to ribonuclease A (RNase A). Based on the systematically docked RNase A-UpA complex resulting from our previous study, we have undertaken a molecular dynamics simulation of the complex with solvent molecules. The molecular dynamics trajectories of this complex are analyzed to provide structural explanations for varied experimental observations on the ligand binding at the B2 subsite of ribonuclease A. The present study suggests that B2 subsite stabilization can be effected by different active site groups, depending on the substrate conformation. Thus when adenosine ribose pucker is O4'-endo, Gln69 and Glu111 form hydrogen-bonding contacts with adenine base, and when it is C2'-endo, Asn71 is the only amino acid residue in direct contact with this base. The latter observation is in support of previous mutagenesis and kinetics studies. Possible roles for the solvent molecules in the binding subsites are described. Furthermore, the substrate conformation is also examined along the simulation pathway to see if any conformer has the properties of a transition state. This study has also helped us to recognize that small but concerted changes in the conformation of the substrate can result in substrate geometry favorable for 2',3' cyclization. The identified geometry is suitable for intraligand proton transfer between 2'-hydroxyl and phosphate oxygen atom. The possibility of intraligand proton transfer as suggested previously and the mode of transfer before the formation of cyclic intermediate during transphosphorylation are discussed.     

Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras

H-Ras, the protein product of the cellular homologue of the Harvey ras oncogene, undergoes a complex series of post-translational modifications that include C-terminal isoprenylation, proteolysis, methylation, and palmitoylation. Palmitoylation has been shown to enhance the transformation efficiency of H-Ras about 10-fold in vivo. A recent study (Magee, A. I., Gutierrez, L., McKay, I. A., Marshall, C. J., and Hall, A. (1987) EMBO J. 6, 3353-3357) has provided strong evidence that the palmitate undergoes a dynamic acylation-deacylation cycle, but details concerning the enzymology of this process and its regulation are lacking. To begin to dissect this event, we have developed an assay for the enzymatic removal of palmitate from [3H]palmitate-labeled H-Ras. This substrate was produced in a baculovirus expression system and was used to purify to homogeneity a novel 37-kDa enzyme from bovine brain cytosol that removes the radiolabeled palmitate. The purified enzyme is sensitive to diethyl pyrocarbonate and insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. Interestingly, the thioesterase recognizes H-Ras as a substrate only when H-Ras is in its native conformation (bound to Mg2+ and guanine nucleotide). The palmitoylated alpha subunits of the heterotrimeric G proteins are also substrates for the enzyme.     

Dissecting cAMP binding domain A in the RIalpha subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit

The two gene-duplicated cAMP binding domains in the regulatory subunits of cAMP dependent protein kinase are each comprised of an A helix, an eight-stranded beta-barrel, and a B and C helix (1). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid screen coupled with deletion analysis, cAMP binding domain A of RI was dissected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit. The minimum stable subdomain required for binding to C in the 1-3 micromolar range is composed of residues 94-169, while residues 236-244, mapped to the C helix of cAMP binding domain A, were defined as a second surface necessary for high affinity (5-10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, serves as a molecular switch for either a cAMP-bound conformation or a C-bound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B.     

Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity

The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.     

Mouse beta-galactoside alpha 2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression

Four types of beta-galactoside alpha 2,3-sialyltransferase (ST3Gal I-IV) have been cloned from several animals, but some contradictory observations regarding their substrate specificities and expression have been reported. Therefore, it is necessary to concurrently analyze the substrate specificities of the four enzymes, of which the source should be one animal. Accordingly, the acceptor substrate specificities and gene expression of mST3Gal I-IV were analyzed. Since we had already cloned ST3Gal I and II, as previously reported (Lee, Y.-C. et al., Eur. J. Biochem., 216, 377-385 (1993); J. Biol. Chem., 269, 10028-10033 (1994)), the cDNAs of ST3Gal III and IV were cloned from mouse cDNA libraries. Each of the four enzymes was expressed in COS-7 cells as a recombinant enzyme fused with protein A, and applied on an IgG-Sepharose gel to eliminate endogenous sialyltransferase activity. ST3Gal I and II showed the highest activity toward Gal beta 1, 3 GalNAc (type III), very low activity toward Gal beta 1,3GlcNAc (type I), but none toward Gal beta 1,4GlcNAc (type II). ST3Gal III and IV exhibited high activity toward the type I and II disaccharides, but very low activity toward the type III one. On the other hand, asialo-GM1 (Gg4Cer) was as good a substrate for ST3Gal I and II as the type III disaccharide, though ST3Gal III and IV hardly utilized glycolipids as substrates, as indicated by in vitro experiments. Northern blot analysis revealed that enzymes of the ST3Gal-family are expressed mainly in a tissue-specific manner. The ST3Gal I gene was strongly expressed in spleen and salivary gland, and weakly in brain, liver, heart, kidney, and thymus. The ST3Gal II gene was strongly expressed in brain, and weakly in colon, thymus, salivary gland, and testis, and developmentally expressed in liver, heart, kidney, and spleen. The ST3Gal III and IV genes were expressed in a wide variety of tissues. These differences in tissue specific expression suggest the expression of each ST3Gal influences the distribution of sialyl-glycoconjugates in vivo.     

Binding of chloromethyl ketone substrate analogues to crystalline papain

Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.     

Site-directed chemical modification of cysteine-scanning mutants as to transmembrane segment II and its flanking regions of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a transmembrane water-filled channel

Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H+ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II. None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation. [14C]N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated. The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A. (1997) J. Biol. Chem. 272, 580-585). However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with [14C]NEM. The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment. Positions reactive to NEM appear periodically in the second half. They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel. The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half. These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway.     

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.