Substrate Recognition Sequence Research Articles

Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2

The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events, such as the activation of viral glycoproteins and bacterial exotoxins. Small, non-toxic and highly active, furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria. In this study, a series of peptide inhibitors were designed and synthesized based on the C-terminal fragment of histone H1.2, which has an inhibitory effect on furin. Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity greatly. The most potent inhibitor, I4, with 14 amino acid residues has a Ki value of 17 nM for furin. Although most of the synthesized peptides were temporary inhibitors, the inhibitor I5, with nine amino acids, retained its full potency, even after a 3 h incubation period with furin at 37 degrees C. These inhibitors may potentially lead to the development of anti-viral and anti-bacterial drug compounds.

Examination of the role of histidine residues proximal to the cleavage site on cathepsin L processing of the Hendra virus fusion protein

The recently emerged Hendra virus is an enveloped, single-stranded, negative-sense RNA virus which is classified as a BSL-4 pathogen. Both the viral attachment (G) and fusion (F) proteins are required for fusion of the viral membrane with the host cell membrane, an obligate early step in infection. Hendra F must undergo proteolytic processing to be fusogenically active, and this unique cleavage event involves trafficking of F to the plasma membrane and subsequent endocytosis and cleavage by the endosomal/lysosomal protease cathepsin L. While cleavage of other paramyxovirus fusion proteins occurs following specific sequences, cathepsin L lacks a specific substrate recognition sequence. As Hendra F cleavage by cathepsin L occurs in acidic endosomal compartments, protonation of residues near the cleavage site could modulate F cleavage. The ectodomain of F contains only two histidine residues, H102 and H372. Mutation of H102, directly upstream of the cleavage site, to an alanine did not significantly alter cleavage or function. Modeling Hendra F on to the prefusion structure of HPIV5 F indicates that H372 is in close proximity to H102, thus local conformational changes driven by histidine protonation could alter the cleavage site. We will therefore examine processing and function of the Hendra F mutants H372A and H102A/H372A to define the possible role of histidine protonation in Hendra F cleavage. This work is supported by grants from the National Institutes of Health and the March of Dimes.

Improvement of Cyclophosphamide Activation by CYP2B6 Mutants: From in Silico to ex Vivo

Cyclophosphamide (CPA) is a chemotherapeutic agent that is primarily activated in the liver by cytochrome P4502B6 (CYP2B6) and then transported to the tumor via blood flow. To prevent deleterious secondary effects, P450-based gene-directed enzyme prodrug therapy (GDEPT) consists of expressing CYP2B6 in tumor cells before CPA treatment. Given the relatively low affinity of CYP2B6 for CPA, the aim of our work was to modify CYP2B6 to increase its catalytic efficiency (V(max)/K(m)) to metabolize CPA into 4'-OH CPA. A molecular model of CYP2B6 was built, and four residues in close contact with the substrate were subjected to mutagenesis. Canine CYP2B11 exhibiting a particularly low K(m) to CPA, the amino acids exclusively present in the CYP2B11 substrate recognition sequences were substituted in human CYP2B6. All mutants (n = 26) were expressed in Saccharomyces cerevisiae and their enzymatic constants (K(m), V(max)) evaluated using CPA as substrate. Five mutants exhibited a 2- to 3-fold higher catalytic efficiency than wild-type CYP2B6. A double mutant, comprising the two most effective mutations, showed a 4-fold increase in K(m)/V(max). Molecular dynamic simulations of several mutants were found to be consistent with the observed modifications in catalytic efficiency. Finally, expression of the CYP2B6 114V/477W double mutant, contrary to wt CYP2B6, allowed switching of a resistant human head and neck cancer cell line (A-253) into a sensitive cell line toward CPA. Thus, we were able to obtain a new efficient CYP2B6 mutant able to metabolize CPA, an important step in the GDEPT strategy for human cancer treatment.

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.

Template‐assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small‐molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22‐residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a K i value of 2.45 × 10−9 m for furin and of 5.60 × 10−7 m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys‐to‐Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N‐terminal and C‐terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1′ being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1′ residue Ser was mutated to d‐Ser or N‐methyl‐Ser. The N‐methyl‐Ser mutant gave rise to a K i value of 4.70 × 10−8 m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d‐Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.

Binding of Two Flaviolin Substrate Molecules, Oxidative Coupling, and Crystal Structure of Streptomyces coelicolor A3(2) Cytochrome P450 158A2

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Engineered delta ribozymes can simultaneously knock down the expression of the genes encoding uracil phosphoribosyltransferase and hypoxanthine–xanthine–guanine phosphoribosyltransferase in Toxoplasma gondii

Engineered delta ribozymes or HDV ribozymes were used as gene expression modulators in Toxoplasma gondii. The substrate recognition sequence of the trans-acting delta ribozyme, which was derived from the self-cleaving motif located on the antigenomic strand of the hepatitis delta virus genome, was modified to target T. gondii transcripts. The mRNA encoding two well-documented genes, namely uracil phosphoribosyltransferase ( UPRT) and hypoxanthine–xanthine–guanine-phosphoribosyltransferase ( HXGPRT) of T. gondii were chosen as the targets for the ribozymes. UPRT and HXGPRT are the operative enzymes of the pyrimidine and purine salvage pathway, respectively. The knockdown of UPRT or HXGPRT expression by the engineered ribozymes resulted in parasites with lower levels of the corresponding transcripts and diminished their abilities to assimilate radioactive pyrimidine or purine analogs. Five out of six engineered ribozymes could cleave their substrates and gave rise to the products, which were detected by primer extension assays. Upon electroporation of individual active ribozymes against the UPRT gene, the uracil incorporation was decreased. Similarly, the ribozymes against HXGPRT caused decreased incorporation of hypoxanthine. When the most active ribozyme against UPRT was used in a combination with the best HXGPRT specific ribozyme, the incorporation of both uracil and hypoxanthine were decreased. Northern blot analysis revealed that the target transcripts were lowered to an undetectable level when specific ribozymes were used, and that the transcripts of the housekeeping gene remained intact. The ribozyme system should thus prove to be effective for the study of gene expression in T. gondii.

Cloning and characterization of two fructokinases from maize

Two cDNA clones ( ZmFRK1 and ZmFRK2) encoding fructokinases (FRKs, E.C. 2.7.1.4) were isolated from maize ( Zea mays L.). They encode polypeptides of 323 and 335 amino acids, respectively. Both of the deduced proteins contain ATP binding motif and putative substrate recognition sequences. They share 73.7% sequence identity, and have high sequence homology with previously isolated FRKs from potato, sugar beet, tomato, and Arabidopsis. The in vitro enzymatic studies of the recombinant ZmFrk1 and ZmFrk2 proteins showed that they both had fructose specific kinase activities, but ZmFrk1 had much higher turnover rate than ZmFrk2. Both ZmFrk1 and ZmFrk2 were inhibited by the substrate fructose although ZmFrk1 was less sensitive. The mRNAs of both ZmFRK1 and ZmFRK2 accumulated to high levels in roots and stems, but were barely detectable in leaves. The expression pattern of the two genes was different during seed development. The transcript of ZmFRK1 only appeared in later stages of development, whereas the ZmFRK2 mRNA was expressed in earlier time. Taken together, our results imply that ZmFRK1 and ZmFRK2 play distinct biological roles during seed development.

Random Mutagenesis of Human Cytochrome P450 2A6 and Screening with Indole Oxidation Products1

Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation <WT but had a kcat for coumarin 7-hydroxylation 13-fold >WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products.

A Bipartite Substrate Recognition Motif for Cyclin-dependent Kinases

Cy or RXL motifs have been previously shown to be cyclin binding motifs found in a wide range of cyclin-Cdk interacting proteins. We report the first kinetic analysis of the contribution of a Cy motif on a substrate to phosphorylation by cyclin-dependent kinases. For both cyclin A-Cdk2 and cyclin E-Cdk2 enzymes, the presence of a Cy motif decreased the K(m(peptide)) 75-120-fold while the k(cat) remained unchanged. The large effect of the Cy motif on the K(m(peptide)) suggests that the Cy motif and (S/T)PX(K/R) together constitute a bipartite substrate recognition sequence for cyclin-dependent kinases. Systematic changes in the length of the linker between the Cy motif and the phosphoacceptor serine suggest that both sites are engaged simultaneously to the cyclin and the Cdk, respectively, and eliminate a "bind and release" mechanism to increase the local concentration of the substrate. PS100, a peptide containing a Cy motif, acts as a competitive inhibitor of cyclin-Cdk complexes with a 15-fold lower K(i) for cyclin E-Cdk2 than for cyclin A-Cdk2. These results provide kinetic proof that a Cy motif located a minimal distance from the SPXK is essential for optimal phosphorylation by Cdks and suggest that small chemicals that mimic the Cy motif would be specific inhibitors of substrate recognition by cyclin-dependent kinases.

Rate-determining steps in phenacetin oxidations by human cytochrome P450 1A2 and selected mutants.

Mutants with altered activities were obtained from random libraries of human cytochrome P450 (P450) 1A2 with the putative substrate recognition sequences (SRS) mutated [Parikh, A., Josephy, P. D., and Guengerich, F. P. (1999) Biochemistry 38, 5283-5289]. Six mutants from SRS 2 (E225I, E225N, F226I, and F226Y) and 4 (D320A and V322A) regions were expressed as oligohistidine-tagged proteins, purified to homogeneity, and used to analyze kinetics of individual steps in the catalytic cycle, to determine which reaction steps have been altered. When the wild-type, E225I, E225N, F226I, F226Y, D320A, and V322A proteins were reconstituted with NADPH-P450 reductase, rates of 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation were in accord with those expected from membrane preparations. Within each assay, the values of k(cat)/K(m) varied by 2-3 orders of magnitude, and in the case of E225I and E225N, these parameters were 7-8-fold higher than for the wild-type enzyme. The coupling efficiency obtained from the rates of product formation and NADPH oxidation was low (<20%) in all enzymes. No correlation was found between activities and several individual steps in the catalytic cycle examined, including substrate binding, reduction kinetics, NADPH oxidation, and H(2)O(2) formation. Quench reactions did not show a burst for either phenacetin O-deethylation or formation of the acetol, a minor product, indicating that rate-determining steps occur prior to product formation. Inter- and intramolecular kinetic deuterium isotope effects for phenacetin O-deethylation were 2-3. In the case of phenacetin acetyl hydroxylation (acetol formation), large isotope effects [(D)k(cat) or (D)(k(cat)/K(m)) > 10] were observed, providing evidence for rate-limiting C-H bond cleavage. We suggest that the very high isotope effect for acetol formation reflects rate-limiting hydrogen atom abstraction; the lower isotope effect for O-deethylation may be a consequence of a 1-electron transfer pathway resulting from the low oxidation potential of the substrate phenacetin. These pre-steady-state, steady-state, and kinetic hydrogen isotope effect studies indicate that the rate-limiting steps are relatively unchanged over an 800-fold range of catalytic activity. We hypothesize that these SRS mutations alter steps leading to the formation of the activated Michaelis complex following the introduction of the first electron.

Crystal structure of the apoptotic suppressor CrmA in its cleaved form

Background: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1β and interleukin-18 (caspase 1). Results: We have determined the structure of a cleaved form of CrmA to 2.26 Å resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of α1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. Conclusions: CrmA exemplifies viral economy, selective pressure having resulted in a ‘minimal’ serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.

Selection and characterization of human cytochrome P450 1A2 mutants with altered catalytic properties.

Random mutagenesis is an approach that has the potential to provide useful information about cytochrome P450 (P450) enzymes but has not been extensively used to date. We applied our previously developed systems for generation of random libraries of human P450 1A2 with the putative substrate recognition sequences mutated (individual residues) and an Escherichia coli genotoxity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). A total of 27 mutants were screened from 6000 clones, a small portion of the library. The sequence changes were identified, and E. coli membranes containing each P450 (with NADPH-P450 reductase expressed using a bicistronic vector) were used to determine kcat and Km values for 7-ethoxyresorufin and phenacetin O-deethylation and the (in vitro) activation of MeIQ with another bacterial genotoxicity system (Salmonella typhimurium umu). Within each assay, the values of kcat/Km varied by 2 orders of magnitude, and in some cases these parameters were 3-4-fold higher than for the native enzyme. The profiles of the mutants varied considerably for the three different reactions. Some of the mutants in the Asp-320 region may be compared with site-directed mutants of rat P450 1A2 already reported, with several differences noted. Other mutants have not been studied before in human P450 1A2 or homologues and are of interest; i.e., all Phe-226 mutants showed considerably reduced activity but Glu-225 mutants had increased catalytic activities. In principle, this approach may be applied to random mutagenesis of any enzyme that converts chemicals to mutagens and can be expressed in bacteria.

Characterization of human sublingual-gland protein kinase by phosphorylation of a peptide related to secreted proteins

Phosphoproteins in human saliva include proline-rich proteins, statherins, histatin I and cystatin SA-III. The presence of phosphate in these proteins is necessary for various functions in the mouth including calcium binding, inhibition of precipitation of calcium phosphate, inhibition of growth of hydroxyapatite crystals and adherence to hydroxyapatite. To elucidate the process of phosphorylation of these proteins, the phosphorylation of a peptide (APRP8) with an amino acid sequence identical to one of the phosphorylated sites in acidic proline-rich proteins by a kinase from the human sublingual gland was investigated. The kinase, which was highly labile, was purified 58-fold by fractionation of sublingual gland homogenate and gel filtration, but the enzyme was inactivated when further purification by chromatographic techniques commonly used for protein kinases was attempted. To compare the enzyme with other kinases, and to obtain information that could be used in its further purification, a characterization was undertaken. The enzyme required 10 MM Mg 2+ for optimum activity, it had a K M of 0.09 mM for ATP and the KM for the peptide substrate APRP8 was 0.42 mM. It was not activated by cAMP or calmodulin, characteristics that are shared with casein kinases and mammary gland kinase. The sublingual kinase as well as casein kinase 2 were inhibited by heparin, but in other respects the two kinases had different properties. While casein kinase 2 is activated by polylysine and has optimal activity in 150 mM KCI, sublingual kinase was inhibited by polylysine and the addition of KCI. Moreover, casein kinase 2 can utilize both ATP and GTP as phosphoryl donors, but GTP was not a substrate for sublingual kinase. The sublingual kinase shared a substrate recognition sequence with mammary gland kinase, but, unlike that kinase, it could not utilize Cat` instead of Mgt 2+. While the sublingual kinase thus shared some properties with both casein kinase 2 and mammary gland kinase, distinct differences were also seen and the relationship to these enzymes remains to be determined. The characterization of the sublingual kinase will be useful in its further purification.

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of CAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by displaying it on the surface of filamentous bacteriophage fd. Phosphorylationin vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-rner peptide library phosphorylated by cAPK was selected with ferric (Fe(3+)) chelation affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Theirin vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe(3+)) chelation affinity chromamraphy to identify the substrate specificity of protein kinase from random peptide library was feasible.

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.

A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40–43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent K m values have been determined to be 15 μM for ATP, 1.2 μM for S6 and 10 μM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n − 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.

Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft

The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coli. Heterologous expression of the protein, followed by refolding in vitro, yields an enzymatically active pro-enzyme which fails to autodigest to form the mature protein. Mutagenesis of the active site of papain to simulate that of PPIV yields a proenzyme which also fails to autoactivate. Complementary mutagenesis of the pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will autocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that such a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity.

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies [Damodaran and Harris (1995, J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R,L]GNPVP[F,G]R[Q,I]XY[G,E]XR(N,A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.

Mutational Analysis of Ca2+-independent Autophosphorylation of Calcium/Calmodulin-dependent Protein Kinase II

Previous studies with synthetic peptides indicate that residues 290-309, corresponding to the calmodulin (CaM)-binding domain of Ca2+/CaM-dependent protein kinase II interact with the catalytic core of the enzyme as a pseudosubstrate (Colbran, R. J., Smith, M. K., Schworer, C. M., Fong, Y. L., and Soderling, T. R. (1989) J. Biol. Chem. 264, 4800-4804). In the present study, we attempted to locate the pseudosubstrate motif by generation or removal of potential substrate recognition sequences (RXXS/T) at selected positions using site-directed mutagenesis. Based on previous results, Arg297, Thr305/306, and Ser314 were selected as key residues. Single mutations such as N294S, K300S, A302R, A309R, and R311A were expressed, purified, and characterized. Several of the mutants exhibited decreased binding of and activation by CaM, not surprising since the mutations were within the CaM-binding domain. None of the mutants exhibited enhanced Ca(2+)-independent kinase activity toward exogenous substrate, but the K300S and N294S mutants showed a significant enhancement in the rate and stoichiometry of 32P incorporation during Ca(2+)-independent autophosphorylation. Using two-dimensional peptide mapping and phosphoamino acid analyses, enhanced phosphorylation of the introduced Ser residue was demonstrated in the K300S mutant but not in the N294S mutant. This specific Ca(2+)-independent autophosphorylation of Ser300 is consistent with the hypothesis that Arg297 may occupy the P (-3) position in a pseudosubstrate autoinhibitory interaction with the catalytic core in the nonactivated state of the kinase.