Logical analysis of the mechanism of protein folding II. The nucleation process

Regions of secondary structure are predicted, without using information about the conformation of the protein itself, and compared with crystallographic assignments for seven proteins of recently published sequence and conformation (Table 1). It is observed in Table 3 that the prediction of helices is good (78.7% for %cor.ass.3), except for proteins having large antiparallel pleated sheets, and the prediction of β-structure is quite good (51.2% for %cor.ass.3) except for helix-rich proteins.The prediction of secondary structure from sequence, and a survey of all protein structures analysed so far by X-ray crystallography, suggest that nuceleation starts in almost all cases from interactions in the medium range between the regions having helical potential (α-candidate) and β-structural potential (β-candidate), which are very close to each other but separated by at least three hydrophilic or neutral residues in four consecutive residues on the polypeptide chain. Predictability of loops or turns is enhanced to 71.3% (%cor.ass.2) from 64.4% by taking into account the contiguous α-β interactions. Such a medium-range interaction is called here a probable nucleus. There are a lot of nuclei in large proteins such as carboxypeptidase Aα, while there exists at least one in small proteins like the trypsin inhibitor, Moreover, such an interaction could be a transitionary state towards a helix-rich protein, and towards a helix-deficient protein having a large antiparallel pleated sheet β-structure as well.The analysis of the relation between probable nuclei with regard to their mutual spatial proximity strongly suggests that the topological pathway of the polypeptide chain in three-dimensional space might be decided by the long-range interactions between an α-candidate and a β-candidate. An empirical rule is observed that almost all parallel pleated sheets are accompanied by helices in their neighbourhood. An accumulation of chemical facts, such as complementation experiments, combinations of disulphide bonds, etc., seems also to be elucidated by the proposed mechanism of protein folding.     

Logical analysis of the mechanism of protein folding III. Prediction of the strong long-range interactions.

It has been found that strong long-range interactions occur in regions having large β-structural potentials. As has been described previously (Nagano, 1974), interactions among regions having both helical and β-structural potentials (αβ-gaβ interactions) are also very important. Accordingly, an idea is presented in this paper that the relative stability of a protein conformation could be estimated by a relatively simple mathematical function of sequence and conformation. The function P(p,q) is called the non-energy part of pseudo-free energy, because minimization of the sum of P(p,q) and energy functions (cf. Levitt, 1974; Warme &; Scheraga, 1974) can be expected to lead to a plausible model of a protein. A merit of the function is that it can help us decide which way to go in manipulating a temporarily built model, e.g. towards a helix-rich protein or towards a β-structure-rich protein. The estimation of P(p,q) as an artificial potential does not use much computer time because only the co-ordinates of the β-carbon atoms (α-carbon atoms if the residue is Gly) are used. It is composed of terms of the long-range interactions P_L and short-range interactions P_S. The term P_L represents the relative strength of helix-helix interactions, helix-β-candidate interactions and β-candidate-β-candidate interactions. It is assumed that both helical and β-structural potentials can be measured as the differences between the predicted function for helix and β-structure, respectively, as defined previously (Nagano, 1973), and the corresponding largest values ever found. A hypothesis that two residues distantly separated in the primary sequence contribute less to the stability of the whole molecule is finally discarded because the true conformation of concanavalin A becomes very unstable compared with its false conformation folded like the main part of subtilisin. The parameters thus determined indicate that the helix-β-candidate interactions are almost as important as the β-candidate-β-candidate interactions. Both helix and loop prediction functions are combined to give the short-range interactions term, P_S, according to whether the region is really helical or not, and to whether it is really looped or not. The function P(p,q) can be used as a criterion for judging whether the predicted conformation is realistic or false, because the parameters can be adjusted to give, within limits, reasonable values of −10 kcal/residue for true conformations and higher than −5 kcal/residue for false conformations.As an application of the present theory of protein folding, the tertiary structure of bacteriophage T4 lysozyme is predicted and presented in Figure 1, prior to the X-ray structure becoming available.     

Theoretical π-π* absorption and circular dichroic spectra of polypeptide β-structures

Absorption and circular dichroic (CD) spectra of the π-π* transition near 200 nm are calculated for poly(Gly), poly(Ala), poly(Abu), and poly(Val) in the β_P (paralle) and β_A (antiparallel) pleated-sheet structures using the dipole interaction model and including interactions among all atoms; optical parameters were obtained from previous studies of related molecules. Most of the calculations are for structures with one or three chains of six residues each. The oscillator strength and splitting of the π-π* modes are found to be affected only to a small extent by variations in side-chain structure and conformation, whereas the CD spectrum is very sensitive to these variations. Poly(Gly) and poly(Ala) β-structures in uniform, planar lattices do not show sufficient rotational strength to account for observed CD spectra. Poly(Abu) and poly(Val) β-structures in uniform, planar lattices show rotational strengths comparable to experiment when χ¹ is near ?60° for β_A-structures or in a broad range near 140° for β_P-structures. Poly(Ala) in uniformly twisted β_A- and β_P-structures or in irregular β_A-structures corresponding to regions of the crystal structure of concanavalin A also show enhanced rotational strengths in the principal π-π* CD band. Absorption and CD spectra calculated for poly(Abu) in uniform β_A- and β_P-structures are compared with experimental data on poly(Lys) in the β-form, assuming side-chain conformations in Abu that maximize the intensity of the principal CD band. The calculations for the β_A-form show the better agreement with experiment for both types of spectra.     

Specific recognition in the tertiary structure of β-sheets of proteins

The frequency of occurrence of nearest neighbour residue pairs on adjacent antiparallel (β_A) and parallel (β_P) strands is obtained from 30 known protein structures. The specificity of interstrand recognition due to such pairing as a factor in the folding of β-sheets is studied by statistical methods. Residues of sufficiently high count for statistical analysis are treated individually while the rest are combined into small groups of similar size, polarity, and/or genetic exchangeability. The hypothesis of specific recognition between individuals and small groups is contrasted with the alternative hypothesis of non-specific recognition between broad classes (hydrophobia, neutral, polar) of residues. A χ² test of pair correlations favours specific recognition against non-specific recognition with a high level of confidence. The largest and most significant correlations are: Ser/Thr (1.9 ± 0.3), Ile/Val (1.7 ± 0.3) and Lys-Arg/Asp-Gln (1.8 ± 0.3) in β_A, and Ile/Leu (1.9 ± 0.4) in β_P. The pair Gly/Gly never occurs in any β-sheet. The specific residue-pair correlations derived here may be useful in statistical prediction methods of protein tertiary structure.     

Use of 5-hydroxy-4H-benzo[1,4]oxazin-3-ones as β2-adrenoceptor agonists

Novel β₂-agonists with a 5-hydroxy-4H-benzo[1,4]oxazin-3-one moiety as head group are described. Systematic chemical variations at the phenethylamine residue of these compounds lead to the discovery of compound 6m as potent, full agonist of the β₂-adrenoceptor with a high β₁/β₂-selectivity. Molecular modeling revealed an interaction between the carboxylic acid group of 6m and a lysine residue (K305) of the β₂-receptor as putative explanation for the high observed selectivity. Further, compound 6m displayed in a guinea pig in vivo model a complete reversal of acetylcholine induced bronchoconstriction which lasted over the complete study time of 5 h.     

Short-range conformational energies,secondary structure propensities,and recognition of correct sequence-structure matches

A statistical analysis of known structures is made for an assessment of the utility of short-range energy considerations. For each type of amino acid, the potentials governing (1) the torsions and bond angle changes of virtual C^α-C^α bonds and (2) the coupling between torsion and bond angle changes are derived. These contribute approximately −2 RT per residue to the stability of native proteins, approximately half of which is due to coupling effects. The torsional potentials for the α-helical states of different residues are verified to be strongly correlated with the free-energy change measurements made upon single-site mutations at solvent-exposed regions. Likewise, a satisfactory correlation is shown between the β-sheet potentials of different amino acids and the scales from free-energy measurements, despite the role of tertiary context in stabilizing β-sheets. Furthermore, there is excellent agreement between our residue-specific potentials for α-helical state and other thermodynamic based scales. Threading experiments performed by using an inverse folding protocol show that 50 of 62 test structures correctly recognize their native sequence on the basis of short-range potentials. The performance is improved to 55, upon simultaneous consideration of short-range potentials and the nonbonded interaction potentials between sequentially distant residues. Interactions between near residues along the primary structure, i.e., the local or short-range interactions, are known to be insufficient, alone, for understanding the tertiary structural preferences of proteins alone. Yet, knowledge of short-range conformational potentials permits rationalizing the secondary structure propensities and aids in the discrimination between correct and incorrect tertiary folds. Proteins 29:292–308, 1997. © 1997 Wiley-Liss, Inc.     

Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site-directed fluorescence quenching

We present a detailed study on the formation of neighboring β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-Strand and β-barrel formations were investigated for the eight-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single-cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring β-strands oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were closer to either the periplasmic turns (named cis-side) or the outer loops (named trans-side) of the strand. W_nC_m OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin label, which is a short-range fluorescence quencher. To monitor the association of neighboring β-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the lipid membrane. Residues in the trans-side of strands β₁, β₂, and β₃, represented by mutants W₁₅C₃₅ (β₁β₂, trans) and W₅₇C₃₅ (β₃β₂, trans), reached close proximity prior to residues in the N(β₁)- and C(β₈)-terminal strands as examined for mutants W₁₅C₁₆₂ (β₁β₈, trans) and W₇C₁₇₀ (β₁β₈, cis). Tryptophan and cysteine converged slightly faster in W₁₅C₁₆₂ (β₁β₈, trans) than in W₇C₁₇₀ (β₁β₈, cis). The last folding step was observed for residues at the cis-ends of strands β₁ and β₂ for the mutant W₇C₄₃ (β₁β₂, cis). The data also demonstrate that the neighboring β-strands associate upon insertion into the hydrophobic core of the lipid bilayer.     

The role of colicin receptors in the uptake of ferrienterochelin by Escherichia coli K-12.

The amino terminal sequences of the 4 caseins synthesized by translation of ovine mammary mRNAs in a wheat germ cell-free system have been investigated by automated Edman degradation. The 3 “calcium-sensitive” caseins (α_s1, α_s2 and β) and κ-casein were synthesized as precaseins with amino terminal hydrophobic extensions of 15 and 21 amino acid residues respectively, resembling “signal peptides” of other secretory proteins. The extra pieces of the 4 caseins, which start with a methionyl residue, end with an alanyl residue which may be one of the signals recognized by the mammary membrane-bound enzyme responsible for the specific cleavage of precaseins. The amino terminal extensions of α_s1, α_s2 and β-caseins show a high degree of homology suggesting that they have derived from a common ancestor.     

Oxygenation properties and oxidation rates of mouse hemoglobins that differ in reactive cysteine content

House mice (genus Mus) harbor extensive allelic variation at two tandemly duplicated genes that encode the β-chain subunits of adult hemoglobin (Hb). Alternative haplotypes differ in the level of sequence divergence between the two β-globin gene duplicates: the Hbb^d and Hbb^p haplotypes harbor two structurally distinct β-globin genes, whereas the Hbb^s haplotype harbors two β-globin duplicates that are identical in sequence. One especially salient difference between the s-type Hbs relative to the d- and p-type Hbs relates to the number of reactive β-chain cysteine residues. In addition to the highly conserved cysteine residue at β93, the d- and p-type Hbs contain an additional reactive cysteine residue at β13. To assess the functional consequences of allelic variation in β-globin cysteine content, we measured O₂-binding properties and H₂O₂-induced oxidation rates of mono- and dicysteinyl β-Hbs from 4 different inbred strains of mice: C57BL/6J, BALB/cJ, MSM/Ms, and CAROLI/EiJ. The experiments revealed that purified Hbs from the various mouse strains did not exhibit substantial variation in O₂-binding properties, but s-type Hb (which contains a single reactive β-chain cysteine residue) was far more readily oxidized to Fe^3 + metHb by H₂O₂ than other mouse Hbs that contain two reactive β-chain cysteine residues. These results suggest that the possession of an additional reactive cysteine residue may protect against metHb formation under oxidizing conditions. The allelic differences in β-globin cysteine content could affect aspects of redox signaling and oxidative/nitrosative stress responses that are mediated by Hb-S-nitrosylation and Hb-S-glutathionylation pathways.     

Role of Glu445 in the substrate binding of β-glucosidase

A previously uncharacterized gene in Neosartorya fischeri was cloned and expressed in Escherichia coli. It was found to encode a β-glucosidase (NfBGL1) distinguishable from other BGLs by its high turnover of p-nitrophenyl β-d-glucopyranoside (pNPG). Molecular determinants for the substrate recognition of NfBGL1 were studied through an initial screening of residues by sequence alignment, a second screening by homology modeling and subsequent site-directed mutagenesis to alter individual screened residues. A conserved amino acid, E445, in the substrate binding pocket of wild-type NfBGL1 was identified as an important residue affecting substrate affinity. Replacement of E445 with amino acids other than aspartate significantly decreased the catalytic efficiency (k_cat/K_m) of NfBGL1 towards pNPG, mainly through decreased binding affinity. This was likely due to the disruption of hydrogen bonding between the substrate and the carboxylate oxygen of the residue at position 445. Density functional theory (DFT) based studies suggested that an acidic amino acid at position 445 raises the substrate affinity of NfBGL1 through hydrogen bonding. The residue E445 is completely conserved indicating that this position can be considered as a crucial determinant for the substrate binding among GHs tested.     

Experimental study on aggregation of model monopeptide molecules: 6. A model for peptide β-structures?

Homo- and hetero-aggregates of monopeptide molecules Bu^tCONHCHRCONHPrⁱ have been studied by ¹H-n.m.r. Two pairing modes of the molecules are found for both types of aggregate, according to the bulkiness of side chains R. Their hydrogen bond patterns are closely related to the interstrand interactions in β_a (antiparallel) and β_p (parallel) sheets of globular proteins. The pairing mode Γ₁₄ of these molecules, similar to that of the residues in β_a-structures, is the most stable disposition if the side-chains are not C^β or C^γ-branched simultaneously. When both side chains are bulky C^β or C^γ-branched groups, the pairing mode Γ₁₂ found in β_p-structures is favoured. This observation is in agreement with the lower content of β_p compared to β_a-structure in globular proteins and the preferential occurrence of C^β and C^γ-branched residues in β_p structures.     

The procaryotic nature of the fatty acid synthetase of developing Carthamus tinctorius L. (safflower) seeds

All component activities involved in the synthesis of fatty acid were detected in crude extracts of developing safflower seeds. The crude extracts were fractionated into three portions by polyethylene glycol (0–5, 5–15, and 15% supernatant). Acetyl-CoA:acyl carrier protein (ACP) transacylase was precipitated about 66% by 5% polyethylene glycol. β-Ketoacyl-ACP reductase and enoyl-ACP reductase I were completely recovered in the 5–15% fraction. β-Ketoacyl-ACP synthetase and enoyl-ACP reductase II were in the 15% supernatant fraction. Malonyl-CoA:ACP transacylase and β-hydroxyacyl-ACP dehydrase were distributed into both fractions of 5–15 and 15% supernatant. When the 5–15% fraction was gel-filtrated on Sephadex G-200 column, β-hydroxyacyl-ACP dehydrase and malonyl-CoA:ACP transacylase were clearly separated from other enzymes, but β-Ketoacyl-ACP reductase and enoyl-ACP reductase I overlapped. However, by hydroxyapatite chromatography, these two reductases were clearly separated. Properties of each enzyme were examined with the samples fractionated by polyethylene glycol. β-Ketoacyl-ACP reductase preferably utilized NADPH (K_m = 16 μM) as hydrogen donor. The K_m for acetoacetyl-ACP was 9 μm. β-Hydroxyacyl-ACP dehydrase had a K_m of 12 μm for crotonyl-ACP. Enoyl-ACP reductase had two forms, I and II, and these two reductases differed from each other as follows: (a) separation by polyethylene glycol (15%) fractionation; (b) the optimum pH; (c) the hydrogen donor specificity; (d) the substrate specificity. From these results, it is concluded that the FAS system of developing safflower seeds was nonassociated and similar to the procaryotic type of Escherichia coli.     

The presence of two GDP-L-fucose: glycoproteine fucosyltransferases in human serum

Two soluble fucosyltransferases have been demonstrated in human serum. One enzyme transfers l-fucose from GDP-l-fucose to the terminal galactose residues of lactose, N-acetyllactosamine, and sialidase-treated α₁-acid glycoprotein, to form the blood group H determinant, α-l-fucosyl-(1 → 2)-β-d-galactosyl-R. The second enzyme transfers fucose to the terminal N-acetylglucosamine residue of sialidase-, β-galactosidase-treated α₁-acid glycoprotein. Serum from a donor with the rare “Bombay” O_h blood group (genotype hh) cannot transfer fucose to terminal galactose residues but has normal levels of the enzyme acting on sialidase-, β-galactosidase-treated α₁-acid glycoprotein. This observation, as well as mixed substrate experiments, demonstrate that the two fucosyltransferase activities are due to two separate enzymes. The GDP-l-fucose:galactoside fucosyltransferase has a pH optimum of 5.5 and the following K_m values: lactose, 31 mm; N-acetyllactosamine, 7.5 mm; sialidase-treated α₁-acid glycoprotein, 6.4 mm. The GDP-l-fucose: N-acetylglucosaminide fucosyltransferase has a pH optimum of 5.0 and a K_m for sialidase-, β-galactosidase-treated α₁-acid glycoprotein of 1.2 mm. The serum GDP-l-fucose: N-acetylglucosaminide fucosyltransferase is distinct from the blood group Lewis-dependent enzyme in milk since the serum enzyme is present in serum from Le (a-b-)donors and since the Le-dependent fucosyltransferase could not be demonstrated in serum from donors carrying the Le gene.     

Studies of the function and location of two cysteines in the beta 2 subunit of tryptophan synthase.

Our findings that the apo β₂ subunit of tryptophan synthase of Escherichia coli is inactivated by the modification of one sulfhydryl residue per monomer by nitrothiocyanobenzoic acid and is reactivated by removal of the CN group indicate that the reactive sulfhydryl residue (SH-I) is essential for catalytic activity. SH-I is shown to be the same residue which was previously found to react with bromoacetylpyridoxamine phosphate and different from a sulfhydryl (SH-II) which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate. The results of partial tryptic digestions of β₂ subunit labeled selectively at SH-I or SH-II show that both sulfhydryl residues are located in the F₁ fragment which also contains the pyridoxal phosphate binding site.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41?000 (FP; residues 1-23; AVGIGALFLGFLGAAGSTMGARSCONH₂) participates in fusion processes underlying virus-cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, ¹²C-Fourier transform infrared (FTIR) spectroscopy indicated the following α-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes∼hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). ¹²C-FTIR spectra also showed disordered FP structures in these environments, along with substantial β-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with ¹³C-carbonyl at multiple sites. Combining these ¹³C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: α-helix (residues 3-16) and random and β-structures (residues 1-2 and residues 17-23). Additional ¹³C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the α-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, ¹³C-FTIR spectroscopy showed α-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel β-structure. FP at low loading in ghosts probably inserts deeply as an α-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, β-sheet. In future studies, ¹³C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

Structural features determining the site specificity of a rat liver cAMP-independent protein kinase.

The Seryl and Threonyl residues affected in α_s1 and in β-caseins by rat liver “casein kinase TS” (a cytosolic cAMP-independent protein kinase) have been identified. All of them, as well as the residues affected by the same enzyme in α_s2-casein are characterized by an acidic group two residues to their C terminus and by being located within predicted β-turns. Several other potential sites of phosphorylation, according to their primary structure, but located outside predicted β-turns, are not significantly labeled by the protein kinase. It seems conceivable therefore that both a definite aminoacid sequence including a critical acidic residue, and the existence of a β-turn are required for the activity of this protein kinase.     

Effect of secondary interaction on the enzymatic activity of trypsin-like enzymes from Streptomyces

The effect of secondary interaction with substrate on the enzymatic activity of trypsin-like enzymes from Streptomyces was studied using Z-Lys-(Ala)_m, Z-(Ala)_nLys-OMe, Z-Lys-X-Ala and Z-X-Lys-OMe (m = 1–4; n = 0–2; X = various amino acid residues) as substrates and a comparison was made with bovine trypsin. These peptides are susceptible to cleavage at the peptide or ester bonds containing the carbonyl group of l-lysine, which enabled determination of the effect of chain-length on either side of the sensitive l-lysine residue in the first two types of peptide, and the effect of side-chains of the amino acid residues immediately neighboring on either side of the sensitive l-lysine residue in the latter two types of peptide. The results indicate that the enzymatic activity of the trypsin-like enzymes are little affected by secondary interaction, similarly as seen with bovine trypsin.     

Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability

The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala)_m, Z-Lys-Leu-(Ala)₂, and Z-(Ala)_n-Lys-(Ala)₃ (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala)₂-Lys-(Ala)₃ was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.