Distinguishing among protein kinases by substrate specificities

In the previous paper, N-methylated peptides were shown to be sensitive probes of substrate conformation within the adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) active site. While it has been shown that other protein kinases will catalyze the phosphorylation of the same peptide sequences as A-kinase, there is as yet little information as to whether the protein kinases differentiate between substrates on the basis of conformation. For this reason, the conformationally restricted N-methylated peptides were used to probe the active site of guanosine cyclic 3',5'-phosphate dependent protein kinase (G-kinase), which is homologous in sequence to [Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry 23, 4207-4218] and which has substrate specificities similar to [Lincoln, T. M., & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3239-3243] those of A-kinase. Although this enzyme appears to bind the peptides in a conformation resembling that of conformation A, it is more able to accommodate backbone methylation than is A-kinase. A peptide substrate at least 700-fold selective for G-kinase over A-kinase was found. Backbone methylation may, therefore, represent a way of making peptide substrates and inhibitors selective for a particular kinase.     

Determination of the substrate specificities of N-acetyl-d-glucosaminyltransferase

Heparan sulfate plays a wide range of physiological and pathological roles. Heparan sulfate consists of glucosamine and glucuronic/iduronic acid repeating disaccharides with various sulfations. Synthesis of structurally defined heparan sulfate oligosaccharides remains a challenge. Access to nonsulfated and unepimerized heparan sulfate backbone structures represents an essential step toward de novo enzymatic synthesis of heparan sulfate. The nonsulfated, unepimerized backbone heparan sulfate is similar to the capsular polysaccharide from Escherichia coli strain K5. The biosynthesis of this capsular polysaccharide involves in N-acetylglucosaminyltransferase (KfiA) and d-glucuronyltransferase (KfiC). In this study, we report the characterization of purified KfiA. KfiA was expressed in a C-terminal six-His fusion protein in BL21 star cells coexpressing chaperone proteins GroEL and GroES. The recombinant KfiA was purified to homogeneity with a Ni-agarose column. The binding affinities of various UDP-sugars for KfiA were determined using isothermal calorimetry titration, indicating that both the N-acetyl group and sugar type may be essential for donor substrates to bind KfiA. Kinetic analysis of KfiA toward different sizes of oligosaccharide revealed that KfiA is less sensitive to the size of the acceptor substrates. The results from this study open a new approach for the synthesis of the heparan sulfate backbone.     

The Campylobacter jejuni thiol peroxidases Tpx and Bcp both contribute to aerotolerance and peroxide-mediated stress resistance but have distinct substrate specificities

The microaerophilic food-borne pathogen Campylobacter jejuni experiences variable oxygen concentrations during its life cycle, especially during transitions between the external environment and the avian or mammalian gut. Single knockout mutations in either one of two related thiol peroxidase genes, tpx and bcp, resulted in normal microaerobic growth (10% [vol/vol] oxygen) but poorer growth than that of the wild type under high-aeration conditions (21% [vol/vol] oxygen). However, a tpx/bcp double mutant had a severe microaerobic growth defect and did not grow at high aeration in shake flasks. Although the single mutant strains were no more sensitive than the wild-type strains in disc diffusion assays with hydrogen peroxide, organic peroxides, superoxide, or nitrosative stress agents, in all cases the double mutant was hypersensitive. Quantitative cell viability and cellular lipid peroxidation assays indicated some increased sensitivity of the single tpx and bcp mutants to peroxide stress. Protein carbonylation studies revealed that the tpx/bcp double mutant had a higher degree of oxygen- and peroxide-induced oxidative protein damage than did either of the single mutants. An analysis of the peroxidase activity of the purified recombinant enzymes showed that, surprisingly, Tpx reduced only hydrogen peroxide as substrate, whereas Bcp also reduced organic peroxides. Immunoblotting of wild-type cell extracts with Tpx- or Bcp-specific antibodies showed increased abundance of both proteins under high aeration compared to that under microaerobic growth conditions. Taken together, the results suggest that Tpx and Bcp are partially redundant antioxidant enzymes that play an important role in protection of C. jejuni against oxygen-induced oxidative stress.     

Nitric oxide is a physiological substrate for mammalian peroxidases

We now show that NO serves as a substrate for multiple members of the mammalian peroxidase superfamily under physiological conditions. Myeloperoxidase (MPO), eosinophil peroxidase, and lactoperoxidase all catalytically consumed NO in the presence of the co-substrate hydrogen peroxide (H(2)O(2)). Near identical rates of NO consumption by the peroxidases were observed in the presence versus absence of plasma levels of Cl(-). Although rates of NO consumption in buffer were accelerated in the presence of a superoxide-generating system, subsequent addition of catalytic levels of a model peroxidase, MPO, to NO-containing solutions resulted in the rapid acceleration of NO consumption. The interaction between NO and compounds I and II of MPO were further investigated during steady-state catalysis by stopped-flow kinetics. NO dramatically influenced the build-up, duration, and decay of steady-state levels of compound II, the rate-limiting intermediate in the classic peroxidase cycle, in both the presence and absence of Cl(-). Collectively, these results suggest that peroxidases may function as a catalytic sink for NO at sites of inflammation, influencing its bioavailability. They also support the potential existence of a complex and interdependent relationship between NO levels and the modulation of steady-state catalysis by peroxidases in vivo.     

Molecular weight differences among potato peroxidases

The isoenzyme of potato peroxidase A5 has MW 105 000; C3—94 000; C4 and C5—56 500 C6—48 500. The isoenzymes retain activity on SDS-gels thereby allowing direct measurement of monomeric MW, even in crude extracts. One of the isoperoxidases showed anomalous behaviour on SDS-electrophoresis.     

Prediction of 492 human protein kinase substrate specificities

Background

Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase.

Results

The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates.

Conclusions

Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the open source PhosphoNET website (http://www.phosphonet.ca).

     

Non-poisson analysis of endonucleases with substrate sequence specificities

Endonucleases with substrate-sequence specificities, such as restriction enzymes, usually cleave small, defined nucleic acid molecules used in enzyme assays at one or only a few sites. The methods in common use for analysis of endonucleases are based on the Poisson distribution. A critical, but usually unstated, assumption of this distribution, however, is that there is a large number of possible reactive sites on each substrate molecule. Thus use of the Poisson distribution may introduce large errors into analysis of such assays. Here we develop a series of appropriate expressions for use in analyzing endonucleases with substrate-sequence specificities.     

Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.     

Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities

As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2

PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate (V(max) of 633 nmol min(-1) mg(-1) and K(m) of 45.5 microM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors.     

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.     

Effects of live coral,epilithic algal communities and substrate type on algal recruitment

The recruitment of algae is a critical process during algal colonization and invasions, including coral-to-algal phase shifts. Although algae are widely assumed to colonize and kill corals, there is very little known about the recruitment dynamics of coral reef algae. This study tested the ability of two dominant macroalgae (Fucales including Sargassum spp. and Lobophora variegata) to settle and grow on healthy coral tissue. The study also explored the effects of interactions with prior occupants, and of abiotic substrate properties (texture, and ceramic and carbonate material). The results indicate that healthy corals were able to prevent attachment or survival of recruits of these macroalgae. This is a significant point, since it suggests that the replacement of corals by algae may often require prior stress or death in the coral tissue. Pre-conditioning of plates at different sites had some effects, but these were relatively minor, whereas there was considerable variation within sites. Some of this variation appeared to be related to the amount of turf algae or crustose coralline algae on the plates. Recruitment was generally, but not always, higher on plates with rougher texture. Overall, this preliminary exploration indicates considerable potential for variability in outcomes of algal colonization, with implications for the dynamics of algal invasions. In particular, the results do not support suggestions that planktonic algal propagules can directly settle on and colonize healthy coral tissue.Communicated by Topic Editor D. Barnes     

A comparison of the substrate specificities of cathepsin D and pseudorenin

Cathepsin D, purified from hog spleen, releases angiotensin I from tetradecapeptide renin substrate and from protein renin substrates purified from hog and human plasma. However, the enzyme does not act on the naturally occurring renin substrate as it exists in plasma nor on purified substrate in the presence of plasma. Cathepsin D releases angiotensin I quantitatively from tetradecapeptide renin substrate and does not further degrade the angiotensin I on prolonged incubation. The pH optimum for cathepsin D prolonged incubation. The pH optimum for cathepsin D acting on tetradecapeptide renin substrate is 4.5, and there is very low activity above pH 7. These properties are very similar to those of pseudorenin, an angiotensin-forming enzyme originally isolated from human kidney, indicating that cathepsin D and pseudorenin may be identical.     

Different substrate specificities of the two DNA ligases of mammalian cells

Mammalian cells contain the DNA ligases I and II. These enzymes show different molecular weights and heat labilities, and antibodies against ligase I do not inhibit ligase II. Here, the nonidentical substrate specificities of the enzymes are described. Under standard reaction conditions DNA ligase I, but not ligase II, catalyzes blunt-end joining of DNA, while ligase II is the only activity that joins oligo(dT) molecules hydrogen-bonded to poly(rA). These differences facilitate the distinction between the two enzymes and should permit further analysis of their functions.     

A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub(F45W)-Xaa) and di-ubiquitin chains (Ub(F45W)-diUb). After removal of the intact substrate by Ni(2+)-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub(F45W) product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.     

Two homologous fungal carbonyl reductases with different substrate specificities

Two homologous fungal short-chain dehydrogenase/reductase (SDR) proteins have been cloned from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus) and expressed in Escherichia coli: trihydroxynaphthalene reductase (3HNR), an enzyme of the melanin biosynthetic pathway that catalyzes the conversion of 1,3,8-trihydroxynaphthalene to vermelone, and 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl), which acts on androgens and estrogens, although its physiological substrate remains to be defined. In the present study, we have compared the structures, specificities to substrates and inhibitors, temperature and pH optima of 3HNR and 17beta-HSDcl. Sequence analysis and homology-built models revealed that these enzymes are highly similar. Both of these enzymes are NADP(H)-preferring reductases and act on steroids at position 17; however, 17beta-HSDcl presented considerably higher initial rates than 3HNR. In vitro, 17beta-HSDcl preferably catalyzed the reduction of 4-estrene-3,17-dione, while the best steroid substrate for 3HNR was 5alpha-androstane-3,17-dione. On the other hand, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), an artificial substrate of 3HNR, was oxidized rapidly by 3HNR, while it was not a substrate for 17beta-HSDcl. Additionally, our data show that tricyclazole, a specific inhibitor of 3HNR, is 100-fold less effective for 17beta-HSDcl inhibition, while flavonoids can inhibit both 3HNR and 17beta-HSDcl. We have also examined the effects of temperature and pH on the oxidation of DDBO by 3HNR and the oxidation of 4-estrene-17beta-ol-3-one by 17beta-HSDcl. The apparent optimal temperature for 3HNR activity was between 25 and 30 degrees C, while it was between 40 and 45 degrees C for 17beta-HSDcl activity. The pH optimum of 3HNR activity was between 8 and 9, and for 17beta-HSDcl, between 7 and 8. Our data show that in spite of high homology and similar backbone structure, differences between 3HNR and 17beta-HSDcl were not only in substrate specificities, but also in temperature and pH optima.