Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound

A new crystal structure of O-acetylserine sulfhydrylase (OASS) has been solved with chloride bound at an allosteric site and sulfate bound at the active site. The bound anions result in a new "inhibited" conformation, that differs from the "open" native or "closed" external aldimine conformations. The allosteric site is located at the OASS dimer interface. The new inhibited structure involves a change in the position of the "moveable domain" (residues 87-131) to a location that differs from that in the open or closed forms. Formation of the external aldimine with substrate is stabilized by interaction of the alpha-carboxyl group of the substrate with a substrate-binding loop that is part of the moveable domain. The inhibited conformation prevents the substrate-binding loop from interacting with the alpha-carboxyl group, and hinders formation of the external Schiff base and thus subsequent chemistry. Chloride may be an analog of sulfide, the physiological inhibitor. Finally, these results suggest that OASS represents a new class of PLP-dependent enzymes that is regulated by small anions.     

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.     

Role of induced fit in enzyme specificity: a molecular forward/reverse switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.     

Structural analyses of covalent enzyme-substrate analog complexes reveal strengths and limitations of de novo enzyme design

We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 Å over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme–inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl–substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.     

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.     

Structural basis for substrate specificities of cellular deoxyribonucleoside kinases.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases.     

Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.     

Topological and dynamical properties of Azurin anchored to a gold substrate as investigated by molecular dynamics simulation

A classical molecular dynamics study of the electron transfer protein azurin, covalently bound to a gold substrate through its native disulphide group, is carried out at full hydration. With the aim of investigating the effects on the protein structure and dynamics as induced by the presence of an electric field, simulations are performed on neutral, positively and negatively charged substrates. A number of parameters, such as the average structure, the root mean square deviations and fluctuations, the intraprotein hydrogen bonds and solvent accessible surface of the protein, are monitored during 10 ns of run. The orientation, the height and the lateral size of the protein, with respect to the substrate are evaluated and compared with the experimental data obtained by scanning probe nanoscopies. The electron transfer properties between the copper redox center and the disulphide bridge bound to the substrate are investigated and briefly discussed.     

The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity

The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.     

Asymmetric binding of steroids to internal and external sites in the glucose carrier of erythrocytes

Steroids inhibit glucose transport in erythrocytes by binding to sites in the carrier which are exposed on both the outer and inner surfaces of the cell membrane. Some steroids are bound almost exclusively at inner sites (androstendione and androstandione), while others are bound about as firmly on one side as the other (corticosterone). Still others exhibit a moderate preference for the internal site (deoxycorticosterone). The inhibition is in all cases competitive with respect to a substrate which is bound at the same surface of the membrane as the inhibitor. However, in experiments on substrate entry, internally bound inhibitors act in an apparently non-competitive fashion, as expected if the carrier model is valid. This behaviour explains the appearance of competitive, non-competitive and mixed inhibitions with different steroids (Lacko, L., Wittke, B. and Geck, P. (1975) J. Cell Physiol. 86, 673–680).     

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes.

Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid 'side' of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

Protein adsorption and leakage in carrier-enzyme systems

The capability of binding enzymes adsorptively to unmodified and silanized silica and glass as well as modified polystyrene carriers was studied for alpha-amylase, beta-amylase, and alpha-chymotrypsin. In most cases a high percentage of protein was bound very firmly under considerable loss of activity. The leakage of protein from the carriers was studied by measuring the intrinsic protein fluorescence on beta-amylase adsorptively bound to aminopropyl silica, aminomethyl, and hexadecylaminomethyl polystyrene. It was compared with the leakage of beta-amylase covalently bound to the same carriers via glutaraldehyde, trichloro-triazine, or benzoquinone. In the absence and in the presence of substrate, at 25 and at 60 degrees C, the leakage rates of the adsorptively bound enzymes were not higher than in the covalently bound systems. The poorest binding stability was found in benzoquinone-coupled beta-amylase derivatives. It is even reduced at higher temperatures, whereas the temperature did not show any remarkable influence on the leakage of the other derivatives. In adsorptively as well as in all the covalently bound systems, the presence of substrate did not promote the protein leakage.     

Structure and catalytic mechanism of the cytosolic D-ribulose-5-phosphate 3-epimerase from rice

Cytosolic D-ribulose-5-phosphate 3-epimerase from rice was crystallized after EDTA treatment and structurally elucidated by X-ray diffraction to 1.9A resolution. A prominent Zn(2+) site at the active center was established in a soaking experiment. The structure was compared with that of the EDTA-treated crystalline enzyme from the chloroplasts of potato plant leaves showing some structural differences, in particular the "closed" state of a strongly conserved mobile loop covering the substrate at its putative binding site. The previous proposal for the active center was confirmed and the most likely substrate binding position and conformation was derived from the locations of the bound zinc and sulfate ions and of three water molecules. Assuming that the bound zinc ion is an integral part of the enzyme, a reaction mechanism involving a well-stabilized cis-enediolate intermediate is suggested.     

Hill coefficient ratios give binding ratios of allosteric enzyme effectors; inhibition, activation, and squatting in deoxycytidylate aminohydrolase (EC 3.5.4.12)

The ratio of the steady-state kinetic Hill coefficients of two different effectors equals (under some rather weak general assumptions) the ratio in which the effectors displace each other from an enzyme. This principle can make implications of experimental allosteric enzyme kinetic data immediately apparent. We can use it to find that one molecule of the allosteric inhibitor of dCMP aminohydrolase, at moderately high effector concentrations, displaces one molecule of substrate, or one molecule of activator, whereas at very high concentrations, one molecule of inhibitor displaces two of substrate. Further use of the principle suggests that substrate, at high concentrations, binds binds to activator sites. However, ratios of substrate, activator, and inhibitor Hill coefficients are incompatible with a simple model of activation in which substrate and activator are bound to the same conformation.     

Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation

CD45 is a transmembrane, two-domain protein-tyrosine phosphatase expressed exclusively in nucleated hematopoietic cells. The Src family kinase, Lck, is a major CD45 substrate in T cells and CD45 dephosphorylation of Lck is important for both T cell development and activation. However, how the substrate specificity of phosphatases such as CD45 is achieved is not well understood. Analysis of the interaction between the cytoplasmic domain of CD45 and its substrate, Lck, revealed that the active, membrane-proximal phosphatase domain of CD45 (CD45-D1) bound to the phosphorylated Lck kinase domain, the SH2 domain, and the unique N-terminal region of Lck. The second, inactive phosphatase domain (CD45-D2) bound only to the kinase domain of Lck. CD45-D2 was unable to bind phosphotyrosine, and its interaction with the kinase domain of Lck was independent of tyrosine phosphorylation. The binding of CD45-D2 was localized to subdomain X (SD10) of Lck. CD45-D2 bound similarly to Src family kinases but bound Csk to a lesser extent and did not bind significantly to the less related kinase, Erk1. CD45 dephosphorylated Lck and Src at similar rates but dephosphorylated Csk and Erk1 at lower rates. Replacement of Erk1 SD10 with that of Lck resulted in the binding of CD45-D2 and the conversion of Erk1 to a more efficient CD45 substrate. This demonstrates a role for CD45-D2 in binding substrate and identifies the SD10 region in Lck as a novel site involved in substrate recognition.     

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase,an Initial Component of the N-End Rule Pathway

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.     

Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme

Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl-CoA dehalogenase, forming a transient covalent link at the 4-position of the benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in total inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al.). The present work investigates complexes formed by D145N dehalogenase and substrate or product. Time-resolved absorption and Raman difference spectroscopic data show that these systems evolve rapidly with time. For the substrate complex, initially the absorption and Raman spectra show the signatures of the substrate bound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resembling the un-ionized product in the active site of wild-type dehalogenase. Similarly, for the product complex, the absorption and Raman spectra initially show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the un-ionized product bound to wild-type enzyme. It is proposed that product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 145 in the major enzyme population. Thus, conversions of substrate to product and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidation occurs with a rate constant of 0.0024 h-1. The rate of product-catalyzed deamidation in Hepes buffer at 22 degrees C was measured by stopped-flow kinetics to be 0.024 s-1, 36000 times faster than the spontaneous process. A feature near 1570 cm-1 could be observed in the early Raman spectra of both substrate and product-enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-like species formed by the attack of the product's 4-O- group on the carbonyl of asparagine's side chain and the subsequent release of ammonia. A reaction scheme is proposed, incorporating these observations.     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.