The role of Glu196 in the environment around the substrate binding site of leucine aminopeptidase from Streptomyces griseus

To investigate the role of Glu196 of leucine aminopeptidase from Streptomyces griseus (SGAP) in SGAP activation by calcium and substrate specificity, we constructed E196X SGAP by saturation mutagenesis. Most mutations led to the abrogation of SGAP activation by calcium, and substitution with Lys led to a marked increase in activity toward Asp-p-nitroanilide (pNA) and a decrease in that toward Lys-pNA. A similar result was obtained from the investigation using non-calcium-activated enzyme from Streptomyces septatus (SSAP). These results indicate that Glu196 of SGAP is associated with the environment around the substrate binding site besides its role in SGAP activation by calcium.     

Change in Substrate Preference of Streptomyces Aminopeptidase through Modification of the Environment around the Substrate Binding Site

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k_cat values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).     

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.     

Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.     

Functional plasticity in the substrate binding site of beta-secretase

The aspartic protease beta-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue beta-peptide, which is the principal biochemical marker of Alzheimer's disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S(1) pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.     

The P4 metal binding site in RNase P RNA affects active site metal affinity through substrate positioning

Although helix P4 in the catalytic domain of the RNase P ribozyme is known to coordinate magnesium ions important for activity, distinguishing between direct and indirect roles in catalysis has been difficult. Here, we provide evidence for an indirect role in catalysis by showing that while the universally conserved bulge of helix P4 is positioned 5 nt downstream of the cleavage site, changes in its structure can still purturb active site metal binding. Because changes in helix P4 also appear to alter its position relative to the pre-tRNA cleavage site, these data suggest that P4 contributes to catalytic metal ion binding through substrate positioning.     

Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine-peptidase: the path to a specific substrate

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-D-alanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent beta-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the beta-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-L-aminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DD-peptidase by at least 3 orders of magnitude (k(cat) = 69 s(-1), K(m) = 7.9 microM, k(cat)/K(m) = 8.7 x 10(6) s(-1) M(-1)); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.     

Chemical modification of dipeptidyl peptidase iv: involvement of an essential tryptophan residue at the substrate binding site

Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.     

The conserved substrate binding site of mitochondrial carriers

Mitochondrial carriers transport nucleotides, co-factors and metabolic intermediates across the inner mitochondrial membrane permeability barrier. They belong to a family of transporters unique to eukaryotes and they differ in structure and transport mechanism from other secondary transporters. The main structural fold consists of a barrel of six transmembrane alpha-helices closed at the matrix side by a salt-bridge network at the bottom of the cavity. The significant sequence conservation in the mitochondrial carrier family suggests that specific recognition of substrates is coupled to a common mechanism of transport. We have identified a common substrate binding site comprising residues that are highly conserved and, as demonstrated by mutagenesis, are essential for function. The binding site explains substrate selectivity, ion coupling and the effects of the membrane potential on transport. The main contact points in the site are related by threefold symmetry like the common structural fold. The substrate is bound at the midpoint of the membrane and may function as a pivot point for the movements of the transmembrane alpha-helices as the carrier changes conformation. The trigger for the translocation event is likely to be the substrate-induced perturbation of the salt bridge network at the bottom of the cavity.     

Novel post-replicative DNA modification in Streptomyces: analysis of the preferred modification site of plasmid pIJ101.

Both Streptomyces lividans and Streptomyces avermitilis have the ability to site specifically modify their DNA, rendering it susceptible to in vitro Tris-dependent double-strand cleavage. We have cloned a 160 bp fragment containing the preferred modification site of plasmid pIJ101 and, employing an in vitro primer extension assay, determined that the modifications occur at guanine residues on either strand separated by 3 bp. These guanines are located within a 6 bp palindromic 'core' sequence. A cloned copy of a 35 bp region of the plasmid containing this core sequence was not recognized by the modifying activity in vivo. To further investigate the nature of the site specificity a set of deletion mutants of the 160 bp sequence were analysed. This revealed that a substantial portion of this sequence is essential for authentic modification. The essential region contains three 13 bp direct repeats, the central one containing the core sequence, while the left-hand and right-hand copies overlap two potential stem-loop structures. Deletion of either left- or right-hand repeat structures abolishes modification within the core sequence, although the left-hand deletion resulted in modification at a secondary site within the right-hand direct repeat. These data support a post-replicative mechanism of modification, underlined by the observation that the modifications are not detected in single-stranded plasmid replication intermediates.     

Elucidation of the substrate binding site of Siah ubiquitin ligase

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two beta strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target.     

The protein substrate binding site of the ubiquitin-protein ligase system

In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.     

Insulin-regulated aminopeptidase: analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine beta-naphthylamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.     

Hydrolysis of thionopeptides by the aminopeptidase from Aeromonas proteolytica: insight into substrate binding

A series of L-leucine aniline analogues were synthesized that contained either a carbonyl or thiocarbonyl as a part of the amide bond. Additionally, the para-position on the phenyl ring of several substrates was altered with various electron-withdrawing or donating groups. The kinetic constants K(m) and k(cat) were determined for the hydrolysis of each of these compounds in the presence of the aminopeptidase from Aeromonas proteolytica (AAP) containing either Zn(II) or Cd(II). The dizinc(II) form of AAP ([ZnZn(AAP)]) was able to cleave both carbonyl and thiocarbonyl containing peptide substrates with similar efficiency. However, the dicadmium(II) form of AAP ([CdCd(AAP)]) was unable to cleave any of the carbonyl-containing compounds tested but was able to cleave the thionopeptide substrates. This is consistent with the borderline hard/soft nature of Zn(II) vs Cd(II). The trends observed in the K(m) values suggest that the oxygen atom of the amide bond directly interacts with the dinuclear active site of AAP. Heterodimetallic forms of AAP that contained one atom of Zn(II) and one of Cd(II) (i.e., [CdZn(AAP)] and [ZnCd(AAP)]) were also prepared. The K(m) values for the thionopeptides substrates are the smallest when Cd(II) is in the first metal binding site, suggesting that substrate binds to the first metal binding site. 1-Phenyl-2-thiourea (PTU) and urea (PU) were also examined to determine the differences between thionopeptide and peptide binding to AAP. PTU and PU were found to be competitive inhibitors of AAP with inhibition constants of 0.24 and 4.6 mM, respectively. The electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] were recorded in the absence and presence of both PU and PTU. Spectral changes were observed for PTU binding to [CoCo(AAP)] and [CoZn(AAP)] but not for [ZnCo(AAP)], while no spectral changes were observed for any of the Co(II)-substituted forms of AAP upon the addition of PU. These data indicate that carbonyl binding occurs only at the first metal binding site. In light of the data presented herein, the substrate binding step in the proposed mechanism of AAP catalyzed peptide hydrolysis can be further refined.     

Identification of substrate binding site of cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis ("charged to alanine") and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.     

Alteration of substrate selectivity through mutation of two arginine residues in the binding site of amadoriase II from Aspergillus sp

Amadoriases I and II are deglycation isoenzymes from Aspergillus sp. of potential relevance for treatment of diabetic complications resulting from excessive protein glycation. Amadoriase II has a preference for anionic substrate with a K(m) of 0.23 and 2.53 mM for fructosylglycine and fructosylpropylamine, respectively. In contrast, the corresponding K(m) values for amadoriase I are 9.75 and 0.023 mM, respectively. Chemical modification of amadoriase II with p-hydroxyphenylglyoxal, a specific arginine-modifying reagent, resulted in an inhibition of enzyme activity toward fructosylglycine, while having less effect on the enzymatic activity toward fructosylpropylamine. Peptide mapping and subsequent mass spectrometry analysis suggest that Arg(112) is one of the sites of p-hydroxyphenylglyoxal modification. Sequence alignment between amadoriase I and amadoriase II revealed that two glutamic acids in amadoriase I align to Arg(112) and Arg(114) in amadoriase II. Site-directed mutation of amadoriase II (R112E, R114E) resulted in reversal of the enzymatic activities toward fructosylglycine and fructosylpropylamine. Our results suggested that Arg(112) and Arg(114) are responsible for the high affinity of amadoriase II toward anionic substrates and determine the substrate selectivity of the enzyme.     

An additional substrate binding site in a bacterial phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.     

Arginine as a substrate binding site in aspartate aminotransferase.

Modification of one or two arginine residues in pig-heart cytoplasmic aspartate aminotransferase with 1,2-cyclohexanedione nearly abolishes its catalytic activity and abolishes its ability to bind dicarboxylic acids. The modification is competitively inhibited by glutaric acid. Modification of the enzyme causes no change in its ability to transaminate alanine, but causes a tenfold increase in the Michaelis constant and a 10⁴ fold decrease in the rate of transamination of aspartate. These results indicate that the binding site for the β-carboxyl group of aspartic acid is an arginine residue.     

Identification of substrate binding site of GroEL minichaperone in solution.

It is difficult to obtain high-resolution structural information on the substrate-binding site of intact GroEL. But minichaperones, domains containing the peptide-binding site of GroEL, do constitute tractable systems for detailed studies. A peptide-binding site was located in crystals of a minichaperone and proposed to constitute a model for substrate-binding. We have now located the substrate binding site of the minichaperone GroEL(193-335) in solution by labelling it at various positions with a fluorescent probe and detecting which positions are perturbed on binding a denatured substrate. The fluorescence of a probe attached to a cysteine residue engineered at position 228 (N terminus of helix H8), 241 (helix H8), 261 (helix H9), or 267 (helix H9) was affected significantly by binding of substrate. But there was little change for a label at positions 193, 212, 217 or 293. The dissociation constants between substrates and minichaperone were evaluated from fluorescence anisotropy assays. The effects of salt and temperature were the same as those with intact GroEL. These results indicate that the region around helices H8 and H9 is the substrate-binding site for the apical domain fragment. Intriguingly, the same site is involved in the binding of GroES. Thus, an important function of GroES in the regulation of the activity of GroEL for substrates is to displace the bound substrate by competing for its binding site.