Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkaliphilic strain of Bacillus.

The nucleotide sequence of the gene for a highly alkaline, low-molecular-mass pectate lyase (Pel-15) from an alkaliphilic Bacillus isolate was determined. It harbored an open reading frame of 672 bp encoding the mature enzyme of 197 amino acids with a predicted molecular mass of 20 924 Da. The deduced amino-acid sequence of the mature enzyme showed very low homology (< 20.4% identity) to those of known pectinolytic enzymes in the large pectate lyase superfamily (the polysaccharide lyase family 1). In an integrally conserved region designated the BF domain, Pel-15 showed a high degree of identity (40.5% to 79.4%) with pectate lyases in the polysaccharide lyase family 3, such as PelA, PelB, PelC, and PelD from Fusarium solani f. sp. pisi, PelB from Erwinia carotovora ssp. carotovora, PelI from E. chrysanthemi, and PelA from a Bacillus strain. By site-directed mutagenesis of the Pel-15 gene, we replaced Lys20 in the N-terminal region, Glu38, Lys41, Glu47, Asp63, His66, Trp78, Asp80, Glu83, Asp84, Lys89, Asp106, Lys107, Asp126, Lys129, and Arg132 in the BF domain, and Arg152, Tyr174, Lys182, and Lys185 in the C-terminal region of the enzyme individually with Ala and/or other amino acids. Consequently, some carboxylate and basic residues selected from Glu38, Asp63, Glu83, Asp106, Lys107, Lys129, and Arg132 were suggested to be involved in catalysis and/or calcium binding. We constructed a chimeric enzyme composed of Ala1 to Tyr105 of Pel-15 in the N-terminal regions, Asp133 to Arg159 of FsPelB in the internal regions, and Gln133 to Tyr197 of Pel-15 in the C-terminal regions. The substituted PelB segment could also express beta-elimination activity in the chimeric molecule, confirming that Pel-15 and PelB share a similar active-site topology.     

Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results. In order to cast some light on the E. coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro. Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity. These three residues were suggested to be involved in the nucleoside binding site. However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity. Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site. Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position. Glu198 was mutated to either Gly, Asp and Gln. All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glu198 both in ribose binding and in interaction with phosphate ions. Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions. Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20%. Both amino acids were suggested to take part in subunit interactions. Our results confirm the structural similarity between E. coli UP and E. coli purine nucleoside phosphorylase (PNP).     

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein

RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The K_m of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.     

Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus

Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases.     

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.     

Alteration of the regiospecificity of human heme oxygenase-1 by unseating of the heme but not disruption of the distal hydrogen bonding network

Heme oxygenase regiospecifically oxidizes heme at the alpha-meso position to give biliverdin IXalpha, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry but partially shifts the oxidation to the beta/delta-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by approximately 90 degrees, causes a slight loss of regiospecificity but combined with the R183E and K18E mutations results primarily in beta/delta-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane.     

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.     

Characterization of the enzymatic properties of the first and second domains of metallocarboxypeptidase D.

Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala >/= Met > Ser, Phe > Tyr > Trp > Thr >/= Gln, Asp, Leu, Gly > Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5. 0-6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. Residues Tyr47, His49, Glu95, Phe96, Pro97, Arg120, and His136 have been predicted to form the active site of restrictocin. In this study, we have individually mutated these amino acids to alanine to probe their role in restrictocin structure and function. The role of Tyr47, His49, Arg120, and His136 was further investigated by making additional mutants. Mutating Arg120 or His136 to alanine or the other amino acids rendered the toxin completely inactive, whereas mutating Glu95 to alanine only partially inactivated the toxin. Mutation of Phe96 and Pro97 to Ala had no effect on the activity of restrictocin. The Tyr47 to alanine mutant was inactive in inhibiting protein synthesis, and had a nonspecific ribonuclease activity on 28S rRNA similar to that shown previously for the His49 to Ala mutant. Unlike the His136 to Ala mutant, the double mutants containing Tyr47 or His49 mutated to alanine along with His136 did not compete with restrictocin to cause a significant reduction in the extent of cleavage of 28S rRNA. In a model of restrictocin and a 29-mer RNA substrate complex, residues Tyr47, His49, Glu95, Arg120, and His136 were found to be near the cleavage site on RNA. It is proposed that in restrictocin Glu95 and His136 are directly involved in catalysis, Arg120 is involved in the stabilization of the enzyme-substrate complex, Tyr47 provides structural stability to the active site, and His49 determines the substrate specificity.     

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.     

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.     

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.     

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.     

Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.     

The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities

CYP17 (17alpha-hydroxylase-17,20-lyase; also P450c17 or P450(17alpha)) catalyses the17alpha-hydroxylation of progestogens and the subsequent acyl-carbon cleavage of the 17alpha-hydroxylated products (lyase activity) in the biosynthesis of androgens. The enzyme also catalyses another type of acyl-carbon cleavage (direct cleavage activity) in which the 17alpha-hydroxylation reaction is by-passed. Human CYP17 is heavily dependent on the presence of the membrane form of cytochrome b(5) for both its lyase and direct cleavage activities. In the present study it was found that substitution of human CYP17 amino acids, Arg(347), Arg(358) and Arg(449), with non-cationic residues, yielded variants that were impaired in the two acyl-carbon bond cleavage activities, quantitatively to the same extent and these were reduced to between 3 and 4% of the wild-type protein. When the arginines were replaced by lysines, the sensitivity to cytochrome b(5) was restored and the acyl-carbon cleavage activities were recovered. All of the human mutant CYP17 proteins displayed wild-type hydroxylase activity, in the absence of cytochrome b(5). The results suggest that the bifurcated cationic charges at Arg(347), Arg(358) and Arg(449) make important contributions to the formation of catalytically competent CYP17.cytochrome b(5) complex. The results support our original proposal that the main role of cytochrome b(5) is to promote protein conformational changes which allow the iron-peroxo anion to form a tetrahedral adduct that fragments to produce the acyl-carbon cleavage products.     

Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.

Previously, using a synthetic peptide strategy, we determined that four distinct regions of human beta-thyrotropin (beta TSH) were responsible for interaction of TSH with the TSH receptor. The most potent of these four regions was the carboxyl-terminus of the subunit, represented by the peptide sequence beta 101-112, which inhibited binding of radiolabeled beta TSH to receptor in radioreceptor assay with an IC50 of approximately 100 microM. In the current studies, we systematically substituted the native amino acids in region beta 101-112 with alanine, and we have determined which residues within this span are important to the binding activity of TSH to its receptor. Substitution of Lys101, Asn103, Tyr104, Cys105, Lys107, and Lys110 with alanine each caused a significant fall in activity as compared to the native sequence, whereas substitution at the remaining positions had little or no effect. Because three of these residues are positively charged at physiologic pH, we hypothesized that this charge may be important to the binding activity of the sequence. We modified the charge characteristics of the region by synthesizing two series of analogs in which the residues identified in the alanine substitution studies were substituted with Arg, D-Lys, and D-Arg at each position. In addition, a series of analogs containing basic residues, either added to or substituted for nonbasic residues in the sequence beta 101-112, was synthesized. Substitution of Arg, D-Lys, and D-Arg for Lys101, Lys107, and Lys110 had little effect on activity; however, inclusion of additional basic residues in the beta 101-112 sequence significantly enhanced the inhibitory activity of the region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation

The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.     

Influential factor contributing to the isoform-specific inhibition by ATP of human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of the nucleotide binding site Lys346

Human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD(+) to NADP(+) upon increasing k(cat,NADP) and decreasing K(m,NADP). Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent malic enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.