Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stoichiometry of the interaction of human plasma alpha 1-proteinase inhibitor with subtilisin BPN'

Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E : I = 1 : 7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr80K) of alpha 1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5 21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact alpha 1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human alpha 1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human alpha 1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.     

Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

Complex of subtilisin BPN' with Streptomyces subtilisin inhibitor. Complex formation concomitant with change in reducibility of disulfide bonds in the inhibitor

Structure of the complex of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' was studied by examining the thermal denaturation and reducibility of disulfide bonds. The denaturation temperature of the complex was significantly higher than that of the enzyme. Two disulfide bonds localized in the inhibitor side were completely reduced in the complex, whereas only one of them was reduced in the free SSI. Gel filtration of the reduced complex solution showed clearly that the main products of reduction of the complex were two peptide fragments of SSI divided at the active site. The resistive disulfide bond in the complexed inhibitor became accessible as a result of a large conformational change due to splitting of the half-reduced inhibitor.     

Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor

The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

Interaction of thiolsubtilisin with Streptomyces subtilisin inhibitor, SSI.

Subtilisin BPN' was chemically converted to thiolsubtilisin and the interaction of this modified enzyme with Streptomyces subtilisin inhibitor (SSI) was examined. SSI competitively inhibited the esterolytic activity of thiolsubtilisin toward p-nitrophenyl acetate with a K1 value of 1.3 X 10(-5) M at pH 7.5 Spectrophotometric analysis of the interaction between SSI and the modified enzyme yielded a Kd value of 4 X 10(-5) M at pH 9.7. These values are about 10(5)-fold greater than the Kd value (less than 10(-9) M at pH 7.5) for the native enzyme. This indicates that the small change in the active site structure of subtilisin (Ser221 to Cys221) leads to a considerable decrease in the binding affinity (by about 6-7 kcal/mol) to SSI.     

Genetic variants of protease inhibitors against fungal protease and α-chymotrypsin from hemolymph of the silkworm,Bombyx mori

Many electrophoretic variants of hemolymph inhibitors of proteases from Aspergillus melleus and pancreatic alpha-chymotrypsin were found using 126 silkworm strains. Six inhibitors of the fungal protease were detected and eight of chymotrypsin; the distribution of inhibitors among Japanese, Chinese, and European races was investigated. Comparison of electrophoretic patterns from F1 hybrids and parents showed that the offspring produce inhibitors of both parental types. Segregation in F2 and backcrossing suggest that the expression of each inhibitor is controlled in most cases by a pair of alleles which are responsible for strong and null bands. Two bands of fungal protease inhibitors C and D were controlled by codominant alleles. These results suggest that polymorphism of hemolymph protease inhibitors in the silkworm would be a useful experimental system for the study of the genetic control of protease inhibitors.     

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.     

Characterization of recombinant C1 inhibitor P1 variants.

Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties. The ability to bind to target proteases, as well as potential substrate-like behavior, was investigated with radioimmunoassays. The P1-Lys variant retained binding capacity toward C1s, plasmin, and kallikrein. In addition, complex formation with C1s was detected for P1-Asn and P1-His. All other P1 substitutions resulted in C1 inhibitor variants that neither complexed with nor were inactivated by C1s, kallikrein, beta-factor XIIa, or plasmin. Electrophoretic studies confirmed that P1-Lys and P1-His can form sodium dodecyl sulfate-resistant complexes with C1s. In contrast, the C1s-P1-Asn complex dissociated upon addition of sodium dodecyl sulfate. Kinetic experiments by the method of progress curves generated association rate constants (kon) with C1s of 4.2 x 10(4) M-1 s-1 for recombinant wild-type C1 inhibitor and 1.7 x 10(4) M-1 s-1 for P1-Lys. For P1-Asn and P1-His, kon was decreased approximately 100-fold. The results from inhibition experiments were compatible with a model of reversible inhibition, although the observed dissociation rate for wild-type C1 inhibitor is too low (1-2 x 10(-6) s-1) to be physiologically relevant. The overall inhibition constant (Ki) was estimated to be 0.03 nM. With P1-Asn, reversible inhibition could be demonstrated directly upon dilution of preformed complexes; the observed dissociation rate constant was 3.2 x 10(-4) s-1; and Ki increased to approximately 380 nM. These findings are discussed in relation to inhibitor specificity and inhibition mechanism.     

Chemical replacement of P1' serine residue at the second reactive site of soybean protease inhibitor C-II

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

Interaction of trypsin-like protease from Streptomyces griseus with an immobilized inhibitor from kidney bean.

An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties.     

Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.