The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Structural principles of the wide substrate specificity of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis> carboxypeptidase T. reconstruction of the carboxypeptidase B primary specificity pocket

Site-directed mutagenesis in the active site of Thermoactinomyces vulgaris carboxypeptidase T (CpT), which is capable of hydrolyzing both hydrophobic and positively charged substrates, resulted in five mutants: CpT1 (A243G), CpT2 (D253G/T255D), CpT3 (A243G/D253G/T255D), CpT4 (G207S/A243G/D253G/T255D), and CpT5 (G207S/A243G/T250A/D253G/T255D). These mutants step-by-step reconstruct the primary specificity pocket of carboxypeptidase B (CpB), which is capable of cleaving only positively charged C-terminal residues. All of the mutants retained the substrate specificity of the wild-type CpT. Based on comparison of three-dimensional structures of CpB and the CpT5 model, it was suggested that the lower affinity of CpT5 for positively charged substrates than the affinity of CpB could be caused by differences in nature and spatial location of Leu247 and Ile247 and of His68 and Asp65 residues in CpT and CpB, respectively, and also in location of the water molecule bound with Ala250. An additional hydrophobic region was detected in the CpT active site formed by Tyr248, Leu247, Leu203, Ala243, CH3-group of Thr250, and CO-groups of Tyr248 and Ala243, which could be responsible for binding hydrophobic substrates. Thus, notwithstanding the considerable structural similarity of CpT and pancreatic carboxypeptidases, the mechanisms underlying their substrate specificities are different.     

Nucleophile specificity of subtilisin in an organic solvent with low water content: investigation via acyl transfer reactions

Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) were used as nucleophiles. The nucleophilic efficiencies of these peptides were characterized by the values of the apparent partition constants, p(app), determined from the HPLC analysis of the reactions. The order of preference for the P'(1) position was estimated to be: Gly > hydrophilic, positively charged > hydrophobic, aromatic > negatively charged > Leu > Pro side chain. For the P'(2) position the order of preference was: Gly > hydrophilic, charged > hydrophobic, aromatic > Pro side chain. The values of p(app) for Gly-Gly-Xaa tripeptides cover a range of only two orders of magnitude, with lower nucleophile efficiency for those with hydrophobic amino acid residues in the P'(3) position. The dipeptide with Pro in P'(1) did not react at all, but a tripeptide having Pro in P'(3) was a very good nucleophile. The negatively charged amino acid residues in the P'(1) position result in very weak nucleophilic behavior, whereas the peptides with Asp or Glu in P'(2) and P'(3) are well accepted. Generally, peptides of the Gly-Xaa or Gly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gly series. The length of the peptide chain or amidation of alpha-carboxyl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and nagarse was observed. (c) 1996 John Wiley & Sons, Inc.     

Reexamination of the role of the leucine/isoleucine zipper residues of phospholamban in inhibition of the Ca2+ pump of cardiac sarcoplasmic reticulum

Phospholamban is a small phosphoprotein inhibitor of the Ca(2+)-pump in cardiac sarcoplasmic reticulum, which shows a distinct oligomeric distribution between monomers and homopentamers that are stabilized through Leu/Ile zipper interactions. A two-faced model of phospholamban inhibition of the Ca(2+)-pump was proposed, in which the Leu/Ile zipper residues located on one face of the transmembrane alpha-helix regulate the pentamer to monomer equilibrium, whereas residues on the other face of the helix bind to and inhibit the pump. Here we tested this two-faced model of phospholamban action by analyzing the functional effects of a new series of Leu/Ile zipper mutants. Pentameric stabilities of the mutants were quantified at different SDS concentrations. We show that several phospholamban mutants with hydrophobic amino acid substitutions at the Leu/Ile zipper region retain the ability to form pentamers but at the same time give the same or even stronger (i.e. L37I-PLB) inhibition of the Ca(2+)-pump than do mutants that are more completely monomeric. Steric constraints prevent the Leu/Ile zipper residues sequestered in the interior of the phospholamban pentamer from binding to the Ca(2+)-pump, leading to the conclusion that the zipper residues access the pump from the phospholamban monomer, which is the active inhibitory species. A modified model of phospholamban transmembrane domain action is proposed, in which the membrane span of the phospholamban monomer maintains contacts with the Ca(2+)-pump around most of its circumference, including residues located in the Leu/Ile zipper region.     

Contributions to the S'-subsite specificity of papain.

The product ratio was analyzed for the papain-catalyzed acyl transfer from the specific acyl donor Mal-Phe-Ala-OEtCl to various nucleophilic amino components, ranging from amino acid amides to tripeptide amides. The data obtained are discussed in terms of binding specificity. From the structure-activity relationships for the S'1-P'1 interaction it follows that only three methyl(ene) groups can be accommodated in the S'1 subsite. Hydrophilic side chains are bound better to S'1 than indicated by their hydrophobicities. Negatively charged amino components are inefficient deacylating agents. However, there was no evidence for electrostatic contributions to the nucleophile binding. Amino components with bulky hydrophobic amino acid residues in the P'2 and in the P'3 position, respectively, are preferentially bound to Mal-Phe-Ala-papain. The results of this study can be applied to the planning of papain-catalyzed peptide synthesis reactions.     

On the effect of mutations in bovine or camel chymosin on the thermodynamics of binding κ‐caseins

Bovine and camel chymosins are aspartic proteases that are used in dairy food manufacturing. Both enzymes catalyze proteolysis of a milk protein, κ‐casein, which helps to initiate milk coagulation. Surprisingly, camel chymosin shows a 70% higher clotting activity than bovine chymosin for bovine milk, while exhibiting only 20% of the unspecific proteolytic activity. By contrast, bovine chymosin is a poor coagulant for camel milk. Although both enzymes are marketed commercially, the disparity in their catalytic activity is not yet well understood at a molecular level, due in part to a lack of atomistic resolution data about the chymosin—κ‐casein complexes. Here, we report computational alanine scanning calculations of all four chymosin—κ‐casein complexes, allowing us to elucidate the influence that individual residues have on binding thermodynamics. Of the 12 sequence differences in the binding sites of bovine and camel chymosin, eight are shown to be particularly important for understanding differences in the binding thermodynamics (Asp112Glu, Lys221Val, Gln242Arg, Gln278Lys. Glu290Asp, His292Asn, Gln294Glu, and Lys295Leu. Residue in bovine chymosin written first). The relative binding free energies of single‐point mutants of chymosin are calculated using the molecular mechanics three dimensional reference interaction site model (MM‐3DRISM). Visualization of the solvent density functions calculated by 3DRISM reveals the difference in solvation of the binding sites of chymosin mutants.     

Horse urinary kallikrein, II. Effect of subsite interactions on its catalytic activity

The effect of secondary-subsite interactions on the catalytic efficiency of horse urinary kallikrein was studied using as substrates oligopeptides and peptidyl-4-nitroanilides with L-Arg at P1. The known secondary specificity of tissue kallikreins for hydrophobic residues at P2 was also demonstrated for horse urinary kallikrein and a higher preference of this enzyme for L-Phe over L-Leu at P2 was evident. Interaction of subsites S3 with D-Pro and D-Phe enhanced the catalytic efficiency but tripeptidyl-4-nitroanilides with acetyl-D-Pro, L-Pro and acetyl-L-Pro at P3 were no better substrates than acetyl-dipeptidyl-4-nitroanilides. The importance of the leaving group for the catalysis was proved by higher kcat/Km values for the peptides in relation to peptidyl-4-nitroanilides containing a common acyl-chain. The low kcat value for the peptide with L-Pro at P'2 stresses the importance of a hydrogen bond between P'2 amide and the carbonyl group at S'2. One L-arginine residue at the leaving group, specially at the P'2 position, decreases the value of the apparent Km. This effect resulting of side-chain interactions with S'2, is impaired by a second L-Arg at P'1.     

Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile–salt–oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I₃₃₀/I₃₅₀ ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.     

Structural and electrostatic properties of the 5-HT3 receptor pore revealed by substituted cysteine accessibility mutagenesis

5-HT(3) receptors are members of the Cys loop family of ligand-gated ion channels. We used the substituted cysteine accessibility method to identify amino acid residues in the channel forming domain, M2 that face the water-accessible surface and to locate their position in the ion conduction pathway. Cysteine was substituted for each residue, one at a time, in the M2 segment (Asp(274)-Asp(298)). 5-Hydroxytryptamine EC(50) values for functional mutants did not vary from wild type (1.4 +/- 0.2 microm) by more than 10-fold, and five mutants were nonfunctional. Covalent modification of the mutant receptors with sulfydryl reagents revealed 11 residues to be water-accessible, with a pattern consistent with an alpha-helix except at Leu(285) and Leu(293). The data suggest that charge selectivity begins at a more cytoplasmic level than Val(291). Modification at some positions (Val(291), Leu(293), Ile(294), Leu(287), and Ser(280)) resulted in channels that were locked open. Reaction rates with accessible cysteines were voltage-dependent at some residues, suggesting that access occurs via the ion channel. Overall the data observed are similar but not identical to that reported for other members of the family and confirms the high degree of structural and functional homology between receptors in the Cys loop receptor family.     

Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography

When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

Residues in the longitudinal, hydrophobic strip-of-helix relate to terminations and crossings of alpha-helices.

An alpha-helix terminates when the virtual extension of its most hydrophobic, longitudinal strip containing Leu, Ile, Val, Phe, and Met lacks those residues. In each of 247 helices a template was fitted to maximize the mean hydrophobicity of positions forming a longitudinal strip-of-helix. The template was then extended into sequences beyond the ends of the helices. Leu, Ile, Val, Phe, and Met occurred in positions in the longitudinal strip-of-helix at an increased frequency (p less than 0.001), but in the first and second positions beyond either end of each true helix, they occurred at the same frequency as for their empirical distribution over all the proteins. Excesses of Asp and Glu were found in the N-terminal loop, and of Arg, His, and Lys in specific positions about the C terminus of helices. The longitudinal hydrophobic strip, the smallest amino acid in that strip, and charged amino acids in that strip, related to rotational and longitudinal orientation of alpha-helices in 15 proteins. Adjacent helices generally crossed through their longitudinal hydrophobic strips. They usually crossed through the smallest residue in the strip. Charged residues, when they occurred in the strips, were excluded from the crossing regions.     

An extensive interaction interface between thrombin and factor V is required for factor V activation

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.     

Critical hydrophobic interactions between phosphorylation and actuator domains of Ca2+-ATPase for hydrolysis of phosphorylated intermediate

Functional roles of seven hydrophobic residues on the interface between the actuator (A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca2+-ATPase were explored by alanine and serine substitutions. The residues examined were Ile179/Leu180/Ile232 on the A domain, Val705/Val726 on the P domain, and Leu119/Tyr122 on the loop linking the A domain and M2 (the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr122 in the crystal structures of Ca2+-ATPase in Ca2+-unbound E2 (unphosphorylated) and E2P (phosphorylated) states but are far apart in those of Ca2+-bound E1 (unphosphorylated) and E1P (phosphorylated) states. The substitution-effects were also compared with those of Ile235 on the A domain/M3 linker and those of T181GE of the A domain, since they are in the immediate vicinity of the Tyr122-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca2+-activated E1P formation from ATP. Substitutions of Ile235 and T181GE blocked the E1P to E2P transition, whereas those in the Tyr122-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile235 and Glu183 also blocked EP hydrolysis. Results indicate that the Tyr122-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu183 properly for hydrolyzing the acylphosphate. Ile235 on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr122-cluster, Ile235, and T181GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca2+.     

Substrate specificities of tissue kallikrein and T-kininogenase: their possible role in kininogen processing.

The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the Km values of T-kininogenase while Val at P'2 increases its kcat values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin

The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.     

A Review of: “Laboratory Techniques in Biochemistry and Molecular Biology Vol. 1, T. S. Work and E. Work (eds.) North Holland Publishing Co., Amsterdam/ American Elsevier Publishing Co. Inc., New York, 1969; 573 + vi pages,indices, hard cover; $29.00”

When calf rennet containing ～ 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorhed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain > 97% pure chymosin. The fraction that passed through unadsorbed was found to contain > 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzvme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. I. Analysis of Leu-rich repeats of human luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

The luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) have an approximately 350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the beta-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these beta strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the beta-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that beta-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Refined structure of porcine pepsinogen at 1.8 A resolution

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.