Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.     

SDS-subtilisin catalyzed synthesis of tetra-peptides containing multifunctional amino acid residues in ethanol

The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction Z---Ala---Ala---Leu---OR+H---Phe---pNA→Z---Ala---Ala---Leu---Phe---pNA, nearly quantitative product yields were observed after 2 h, regardless of whether an activated (R=CH₃, p-C₆H₅Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P₁-position. Tetra-peptides of general formula Z---Ala---Ala---P₁---P₁′---pNA, where P₁=Glu, Asp, Lys, Arg or His and P₁′=Phe, Arg or Glu have been obtained in good yield.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

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.     

Specificity of memapsin 1 and its implications on the design of memapsin 2 (beta-secretase) inhibitor selectivity

Memapsin 1 is closely homologous to memapsin 2 (BACE), or beta-secretase, whose action on beta-amyloid precursor protein (APP) leads to the production of beta-amyloid (A beta) peptide and the progression of Alzheimer's disease. Memapsin 2 is a current target for the development of inhibitor drugs to treat Alzheimer's disease. Although memapsin 1 hydrolyzes the beta-secretase site of APP, it is not significantly present in the brain, and no direct evidence links it to Alzheimer's disease. We report here the residue specificity of eight memapsin 1 subsites. In substrate positions P(4), P(3), P(2), P(1), P(1)', P(2)', P(3)', and P(4)', the most preferred residues are Glu, Leu, Asn, Phe, Met, Ile, Phe, and Trp, respectively, while the second preferred residues are Gln, Ile, Asp, Leu, Leu, Val, Trp, and Phe, respectively. Other less preferred residues can also be accommodated in these subsites of memapsin 1. Despite the broad specificity, these residue preferences are strikingly similar to those of human memapsin 2 [Turner et al. (2001) Biochemistry 40, 10001-10006] and thus pose a serious problem to the design of differentially selective inhibitors capable of inhibiting memapsin 2. This difficulty was confirmed by the finding that several potent memapsin 2 inhibitors effectively inhibited memapsin 1 as well. Several possible approaches to overcome this problem are discussed.     

Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Identification of mouse MD-2 residues important for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 antibodies,and for conferring LPS and taxol responsiveness on mouse TLR4 by alanine-scanning mutagenesis

The expression of MD-2, which associates with Toll-like receptor (TLR) 4 on the cell surface, confers LPS and LPS-mimetic Taxol responsiveness on TLR4. Alanine-scanning mutagenesis was performed to identify the mouse MD-2 residues important for conferring LPS and Taxol responsiveness on mouse TLR4, and for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 Ab MTS510. Single alanine mutations were introduced into mouse MD-2 (residues 17-160), and the mutants were expressed in a human cell line expressing mouse TLR4. Mouse MD-2 mutants, in which a single alanine mutation was introduced at Cys37, Leu71, Leu78, Cys95, Tyr102, Cys105, Glu111, Val113, Ile117, Pro118, Phe119, Glu136, Ile138, Leu146, Cys148, or Thr152, showed dramatically reduced ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, and the mutants also showed reduced ability to confer LPS and Taxol responsiveness. In contrast, mouse MD-2 mutants, in which a single alanine mutation was introduced at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154, and His155 showed normal ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, but their ability to confer LPS and Taxol responsiveness was apparently reduced. These results suggest that the ability of MD-2 to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510 is essential for conferring LPS and Taxol responsiveness on TLR4, but not sufficient. In addition, the required residues at codon numbers 34, 85, 101, 122, and 153 for the ability of mouse MD-2 to confer LPS responsiveness are partly different from those for Taxol responsiveness.     

Bindings of axial ligands to cytochrome P-450d mutants: a difference absorption spectral study

By site-directed mutagenesis, we made several cytochrome P-450d (P-450d) mutants as follows: Asn310Phe (D13), Ile312Leu (D14), Glu318Asp (D15), Val320Ile (D16), Phe325Thr (D19), Asn310Phe,Ile312Leu (M6), Glu318Asp,Val320Ile (M7), Phe325Thr, Glu318Asp (M3). This region (Asn-310-Phe-325) is supposed to be located in the distal helix above the heme plane in P-450d, being conjectured from the structure of P-450cam. We studied Soret spectral changes of those mutants by adding several axial ligands such as aniline, pyridine, metyrapone, 2-phenylimidazole and 4-phenylimidazole. Binding constants (Kb) of aniline and pyridine to the single and double mutants were higher than those to the wild type by 2-10-times. The double mutations did not additively increase the Kb values compared with those to the single mutants. In contrast, Kb value (1.0.10(5) M-1) of metyrapone to the double mutant M3 was much higher than that (2.0.10(3) M-1) of the wild type and those of the single mutants, D15 (4.5.10(4) M-1) and D19 (1.6.10(4) M-1). The increased affinity of metyrapone to the mutant M3 may be attributed to an interaction of the hydrophobic group of metyrapone with nearby hydrophobic group(s) produced cooperatively by the double mutation of P-450d. Kb values of 2-phenylimidazole and 4-phenylimidazole to the mutant M3 were also the highest among those of the mutants and the wild type. Therefore, it was suggested that this region (from Asn-310 to Phe-325) must be located at the distal region of the heme moiety and form, at least, a substrate-binding region of membrane-bound P-450d.     

The hydrophobic residues phenylalanine 184 and leucine 187 in the type-1 parathyroid hormone (PTH) receptor functionally interact with the amino-terminal portion of PTH-(1-34).

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH(2)-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182-190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe(184), Arg(186), Leu(187), and Ile(190) as important determinants of maximum binding of (125)I-labeled bovine PTH-(1-34) and (125)I-labeled bovine PTH-(3-34) and determinants of responsiveness to the NH(2)-terminal analog, PTH-(1-14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu(22), Trp(23)]PTHrP-(15-36) to inhibit the cAMP response induced by PTH-(1-34). At Phe(184) and Leu(187), hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1-34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe(184) and Leu(187) in the PTH-1 receptor plays an important role in determining functional interaction with the 3-14 portion of PTH.     

The 10th and 11th residues of short length N-terminal PTH(1-12) analogue are important for its optimum potency.

The 10th and 11th residues of parathyroid hormone PTH(1-12) analogues were substituted to study the structure and function of PTH analogues. The substitution of Ala(10) of [Ala(3,10,12)(Leu(7)/Phe(7))Arg(11)]rPTH(1-12)NH(2) with Glu(10) and/or the Arg(11) with Ile(11) markedly decreased cAMP generating activity. Data from circular dichroism (CD) and the nuclear magnetic resonance (NMR) structural analysis of [Ala(3,10,12)(Leu(7)/Phe(7))Arg(11)]rPTH(1-12)NH(2) revealed tight alpha-helical structures, while the Glu(10) and/or Ile(11) substituted analogues showed unstable alpha-helical structures. We conclude that 10th and 11th residues are important for stabilizing its helical conformation and that destabilization of the alpha-helical structure, induced by substituting the above residues, remarkably affect its biological potency.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

Calcium binding by troponin-C. A proton magnetic resonance study.

The conformational changes induced by the binding of Ca(II) to rabbit skeletal muscle troponin-C (TNC) have been followed by proton magnetic resonance spectroscopy. Ca(II)-free TNC (apo-TNC) contains definite ordered regions. Ca(II) titration of the high affinity sites (cf. Potter , Gergely, 1975) causes a large folding of the backbone, some of which involves refolding of an ordered region(s) and changes in several side-chains e.g. Glu, Asp and Phe. Titration of the low affinity sites does not alter the backbone but leads to changes among hydrophobic side-chains (one or more Val, Leu, Ile; two or more Phe, Glu and Asp) that define an ordered region(s) of apo-TNC. The rate constants for the conformation changes of the low and high affinity sites are approximately 10 s^?1 and < 20 s^?1, respectively. Final stages of the titration include a downfield shifted methyl group (likely Ile) and a Phe residue. The thermal stabilities of apo-TNC, TNC · Ca₂(II) and native TNC were compared. It was concluded that Ca(II) binding by the two high affinity sites both directs and stabilizes much of the structure. The role of the changes of the low affinity sites, which are thought to activate contraction, are briefly discussed.     

Responses of the pancreatic digestive enzyme secretion to various combinations of amino acids and cholecystokinin in chicks (Gallus domesticus)

1. Effect of amino acid administration on pancreatic secretion of digestive enzymes, amylase, trypsinogen and chymotrypsinogen was studied after wing vein injection of an amino acid (AAs) mixture (Thr, Lys, Phe, Leu, Ile, Glu, Val, His, and Met) or combinations of selected amino acids, i.e. Thr + Phe + Ile, Thr + Phe, Thr + Ile or Phe + Ile, in the presence of cholecystokinin (CCK) in chicks. 2. Time course changes of enzyme output were similar in all treatment groups having a peak within 10-30 min, except for Phe + Ile that resulted in delayed induction of the enzyme release as shown by significant increases in the last 20 min compared with those in the rest. 3. When increases in enzyme outputs for the first 30 min were compared, it was shown that the three enzyme responses brought about by the administration of the AAs mixture was almost entirely accounted for by the combined injection of Thr + Phe. 4. Neither Thr + Ile nor Phe + Ile was as effective as Thr + Phe in inducing the output of these pancreatic enzymes. 5. The present results suggest that Thr and Phe may have a specific regulatory role in the secretion of pancreatic digestive enzymes in chicks when administered simultaneously.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

Studies on peptides. LXXXI. Application of a new arginine derivative, NG-mesitylene-2-sulfonylarginine, to the synthesis of substance P and neurotensin.

A new devised arginine derivative, NG-mesitylene-2-sulfonylarginine, Arg(Mts), was employed for the synthesis of hypothalamic substance P and neurotensin. The former was obtained in 74% yield by treatment of the protected undecapeptide amide, Z - Arg(Mts) - Pro - Lys(Z) - Pro - Gln - Gln - Phe - Phe - Gly - Leu - Met(O)-NH2, with methanesulfonic acid in the presence of anisole followed by reduction of the sulfoxide with 2-mercaptoethanol. The latter was obtained in 54% yield by the similar treatment of the protected tridecapeptide ester, Z - Pyr - Leu - Tyr - Glu(OBzl) - Asn - Lys(Z) - Pro - Arg(Mts) - Arg(Mts) - Pro - Tyr - Ile - Leu - OBzl, with methanesulfonic acid. As scavenger, a mixture of anisole-thioanisole-o-cresol (1:1:1, by vol.) was employed to suppress the side reaction, O-mesitylene-2-sulfonation of the Tyr residue.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

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.     

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).     

Novel inhibitor for prolyl aminopeptidase from Serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor

Prolyl aminopeptidase from Serratia marcescens hydrolyzed x-beta-naphthylamides (x=prolyl, alanyl, sarcosinyl, L-alpha-aminobutylyl, and norvalyl), which suggested that the enzyme has a pocket for a five-member ring. Based on the substrate specificity, novel inhibitors of Pro, Ala, and Sar having 2-tert-butyl-[1,3,4]oxadiazole (TBODA) were synthesized. The K(i) value of Pro-TBODA, Ala-TBODA, and Sar-TBODA was 0.5 microM, 1.6 microM, and 12mM, respectively. The crystal structure of enzyme-Pro-TBODA complex was determined. Pro-TBODA was located at the active site. Four electrostatic interactions were located between the enzyme and the amino group of Pro inhibitors (Glu204:0E1-N:Inh, Glu204:0E2-N:Inh, Glu232:0E1-N:Inh, and Gly46:O-N:Inh), and the residue of the inhibitors was inserted into the hydrophobic pocket composed of Phe139, Leu141, Leu146, Tyr149, Tyr150, and Phe236. The roles of Phe139, Tyr149, and Phe236 in the hydrophobic pocket and Glu204 and Glu232 in the electrostatic interactions were confirmed by site-directed mutagenesis, which indicated that the molecular recognition of proline is achieved through four electrostatic interactions and an insertion in the hydrophobic pocket of the enzyme.