Lys40 but not Arg143 influences selectivity of angiotensin conversion by human alpha-chymase

Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys40 or Arg143 accounts for the human enzyme's marked preference for Phe8 over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys40 or Arg143 to neutral residues. Lys40 was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-chymase 1. The Lys40Ala mutant is a dog-like enzyme retaining strong preference for Phe8 but with Tyr4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg143Gln mutant, contrary to prediction, remains highly Phe8-selective. Therefore, Lys40, but not Arg143, contributes to human chymase's remarkable preference for AT II generation over destruction.     

Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].     

Site-directed mutagenesis of human prorenin. Substitution of three arginine residues in the propeptide with glutamine residues yields active prorenin

Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.     

Contribution of basic residues of the autolysis loop to the substrate and inhibitor specificity of factor IXa

The autolysis loop (residues 143-154 in chymotrypsinogen numbering) plays a pivotal role in determining the macromolecular substrate and inhibitor specificity of coagulation proteases. This loop in factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150) whose contribution to the protease specificity of factor IXa has not been studied. Here, we substituted these residues individually with Ala in Gla-domainless forms of recombinant factor IX expressed in mammalian cells. All mutants exhibited normal amidolytic activities toward a FIXa-specific chromogenic substrate. However, Arg143 and Lys147 mutants showed a approximately 3- to 6-fold impairment in FX activation, whereas the Arg150 mutant activated factor X normally both in the absence and presence of factor VIIIa. By contrast, Arg143 and Lys147 mutants reacted normally with antithrombin (AT) in both the absence and presence of the cofactor, heparin. However, the reactivity of the Arg150 mutant with AT was impaired 6.6-fold in the absence of heparin and 33- to 70-fold in the presence of pentasaccharide and full-length heparins. These results suggest that Arg143 and Lys147 of the autolysis loop are recognition sites for FX independent of factor VIIIa, and Arg150 is a specific recognition site for AT that can effectively interact with AT only if the serpin is in the heparin-activated conformation.     

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

Role of basic residues of the autolysis loop in the catalytic function of factor Xa

The autolysis loop of factor Xa (fXa) has four basic residues (Arg(143), Lys(147), Arg(150), and Arg(154)) whose contribution to protease specificity of fXa has not been examined. Here, we substituted these basic residues individually with Ala in the fX cDNA and expressed them in mammalian cells using a novel expression/purification vector system. Following purification to homogeneity and activation by the factor X activator from Russell viper venom, the mutants were characterized with respect to their ability to assemble into the prothrombinase complex to activate prothrombin and interact with target plasma fXa inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin. We show that all mutants interacted with factor Va with normal affinities and exhibited wild-type-like prothrombinase activities toward prothrombin. Lys(147) and Arg(154) mutants were inhibited by TFPI approximately 2-fold slower than wild type; however, both Arg(143) and Arg(150) mutants were inhibited normally by the inhibitor. The reactivities of Arg(143) and Lys(147) mutants were improved approximately 2-fold with antithrombin in the absence but not in the presence of heparin cofactors. On the other hand, the pentasaccharide-catalyzed reactivity of antithrombin with the Arg(150) mutant was impaired by an order of magnitude. These results suggest that Arg(150) of the autolysis loop may specifically interact with the activated conformation of antithrombin.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GAATTC-

According to the X-ray structure analysis of an EcoRI-oligodeoxynucleotide complex [McClarin et al. (1986) Science 234, 1526], sequence specificity is mediated by 12 hydrogen bonds, 6 from each of the two identical subunits of the dimeric enzyme to the recognition site -GAATTC-: Arg200 forms two hydrogen bonds with guanine, while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. Changing the hydrogen-bonding potential at the recognition site without perturbing the rest of the interface should lead to the recognition of degenerate sequences [Rosenberg et al. (1987) in Protein Engineering (Oxender, D. L., & Fox, C. F., Eds.) pp 237-250, Liss, New York]. We have shown previously that replacing Glu144 by Gln and Arg145 by Lys affects the activity of the enzyme, not, however, its specificity [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. We show now that also the mutation of Arg200 to Lys, the double mutation Glu144Arg145 to GlnLys, and the triple mutation Glu144Arg145Arg200 to GlnLysLys do not lead to a detectable degeneracy of the specificity of cleavage by EcoRI but significantly impair the catalytic activity of this enzyme. A detailed analysis of the steady-state kinetics of cleavage of pUC8 DNA and a tridecadeoxynucleotide substrate demonstrates that the reduction in activity for all DNA binding site mutants investigated so far is mainly due to a decrease in kcat, with the exception of the Arg200 to Lys mutant, which is only impaired in its KM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: relevance of arginine 70 for catalysis

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg70, whose guanidinium group is close to the PEP carboxyl group. Arg70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg70Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.     

Site-Directed Mutagenesis of Cytochrome P450scc (CYP11A1). Effect of Lysine Residue Substitution on Its Structural and Functional Properties

Our previous chemical modification and cross-linking studies identified some positively charged amino acid residues of cytochrome P450scc that may be important for its interaction with adrenodoxin and for its functional activity. The present study was undertaken to further evaluate the role of these residues in the interaction of cytochrome P450scc with adrenodoxin using site-directed mutagenesis. Six cytochrome P450scc mutants containing replacements of the surface-exposed positively charged residues (Lys103Gln, Lys110Gln, Lys145Gln, Lys394Gln, Lys403Gln, and Lys405Gln) were expressed in E. coli cells, purified as a substrate-bound high-spin form, and characterized as compared to the wild-type protein. The replacement of the surface Lys residues does not dramatically change the protein folding or the heme pocket environment as judged from limited proteolysis and spectral studies of the cytochrome P450 mutants. The replacement of Lys in the N-terminal sequence of P450scc does not dramatically affect the activity of the heme protein. However, mutant Lys405Gln revealed rather dramatic loss of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in a reconstituted system, and apparent dissociation constant for adrenodoxin binding. The present results, together with previous findings, suggest that the changes in functional activity of mutant Lys405Gln may reflect the direct participation of this amino acid residue in the electrostatic interaction of cytochrome P450scc with its physiological partner, adrenodoxin.     

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replica plate screen facilitated the isolation of mutant subtilisins from Bacillus amyloliquefaciens that had altered autolytic stability under alkaline conditions. Out of about 4000 clones screened, approximately 70 produced subtilisins with reduced stability (negatives). Two clones produced a more stable subtilisin (positives) and were identified as having a single mutation, either Ile107Val or Lys213Arg (the wild-type amino acid is followed by the codon position and the mutant amino acid). One of the negative mutants, Met50Val, was at a site where other homologous subtilisins contained a Phe. When the Met50Phe mutation was introduced into the B. amyloliquefaciens gene, the mutant subtilisin was more alkaline stable. The double mutant (Ile107Val/Lys213Arg) was more stable than the isolated single mutant parents. The triple mutant (Met50Phe/Ile107Val/Lys213Arg) was even more stable than Ile107Val/Lys213Arg (up to two times the autolytic half-time of wild-type at pH 12). These studies demonstrate the feasibility for improving the alkaline stability of proteins by random mutagenesis and identifying potential sites where substitutions from homologous proteins can improve alkaline stability.     

Species-specific inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid

IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetuscontains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gln, respectively, in the human type 2 enzyme. We characterized three mutants of T. foetusIMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K(i) for MPA inhibition and a 8-13-fold increase in the K(m) values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K(i) for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure

Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

Species specificity of amidine-based urokinase inhibitors

Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.     

Ligand Binding and Modulation of Cyclic AMP Levels Depend on the Chemical Nature of Residue 192 of the Human Cannabinoid Receptor 1

Abstract: The human cannabinoid receptor associated with the CNS (CB1) binds Δ⁹-tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein-coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys¹⁹² of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP-55,940 and the aminoalkylindole WIN 55,212-2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP-55,940, whereas WIN 55,212-2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand-induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC₅₀ value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212-2, an order of magnitude difference was observed for the EC₅₀ for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP-55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212-2 binding, the data suggest a role for Lys¹⁹² in WIN 55,212-2-induced receptor activation.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.