Structural basis of inhibition revealed by a 1:2 complex of the two-headed tomato inhibitor-II and subtilisin Carlsberg

Multidomain proteinase inhibitors play critical roles in the defense of plants against predation by a wide range of pests. Despite a wealth of structural information on proteinase-single domain inhibitor interactions, the structural basis of inhibition by multidomain proteinase inhibitors remains poorly understood. Here we report the 2.5-A resolution crystal structure of the two-headed tomato inhibitor-II (TI-II) in complex with two molecules of subtilisin Carlsberg; it reveals how a multidomain inhibitor from the Potato II family of proteinase inhibitors can bind to and simultaneously inhibit two enzyme molecules within a single ternary complex. The N terminus of TI-II initiates the folding of Domain I (Lys-1 to Cys-15 and Pro-84 to Met-123) and then completes Domain II (Ile-26 to Pro-74) before coming back to complete the rest of Domain I (Pro-84 to Met-123). The two domains of TI-II adopt a similar fold and are arranged in an extended configuration that presents two reactive site loops at the opposite ends of the inhibitor molecule. Each subtilisin molecule interacts with a reactive site loop of TI-II through the standard, canonical binding mode. Remarkably, a significant distortion of the active site of subtilisin is induced by the presence of phenylalanine in the P1 position of reactive site loop II of TI-II. The structure of the TI-II.(subtilisin)2 complex provides a molecular framework for understanding how multiple inhibitory domains in a single Potato II type proteinase inhibitor molecule from the Potato II family act to inhibit proteolytic enzymes.     

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).     

Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.     

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.     

Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism

The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.     

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.     

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.     

A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.     

Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor.

Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'.     

Purification and characterization of the trypsin inhibitor from Cucurbita pepo var. patissonina fruits

Three trypsin inhibitor fractions were found in white bush fruits (Cucurbita pepo L. var. patissonina). One of them, CPPTI-fIII, was purified to homogeneity by means of affinity and ion exchange chromatography. It is a cysteine-poor protein with an approximate Mr of 21 000. The inhibitor contains arginine at position P1 of the reactive site and inhibits bovine trypsin, hog pancreatic kallikrein and subtilisin. This inhibitor differs from the inhibitors of white bush dormant seeds, CPPTI-I and CPPTI-II, in its amino-acid composition, molecular mass, amino-acid residue at position P1 of the reactive site and inhibition spectrum.     

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.     

Alteration of inhibitory properties of Pleurotus ostreatus proteinase A inhibitor 1 by mutation of its C-terminal region

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins. In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene. The wild-type POIA1 was found to inhibit subtilisin BPN' with an inhibitor constant (K(i)) of 3.2 x 10(-9) M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN' was used as a target protease. Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN'. The mutated POIA1 inhibited subtilisin BPN' with a K(i) value of 2.8 x 10(-11) M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease. These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation. Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region.     

Crystal structure of subtilisin complexed with its trapped substrateStreptomyces subtilisin inhibitor—Protein-protein interaction and evolution of serine proteinases and their proteinaceous inhibitors

The complex of a bacterial alkaline serine proteinase, subtilisin BPN’, with its proteinaceous inhibitorStreptomyces subtilisin inhibitor is unique in several respects, compared with other similar complexes containing serine proteinases of trypsin family. In addition to the usual antiparallelβ-sheet involving P₁-P₃ residues of the inhibitor, P₄-P₆ residues form antiparallelβ-sheet with a previously unnoticed chain segment (the ‘S4-6 site’) of subtilisin. The ‘S4-6 site’ does not exist in serine proteinases of trypsin family, whether of mammalian or microbial origin. Global induced-fit movement seems to occur on the ‘trapped substrate’Streptomyces subtilisin inhibitor: a channel-like structure in SSI remote from the contact region becomes about 2 Å wider upon complexing with subtilisin. Main role of the secondary contact region ofStreptomyces subtilisin inhibitor seems to support the reactive site loop (primary contact region). Steric homology for the two contact regions is so high between the inhibitors ofStreptomyces subtilisin inhibitor family and those of pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family that it seems to favour a divergent evolution and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forwarded by Doolittle(Nature (London),272, 581, 1978).     

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.     

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.     

Mechanisms of temporary inhibition in Streptomyces subtilisin inhibitor induced by an amino acid substitution, tryptophan 86 replaced by histidine

Just one amino acid substitution (Trp86 replaced by His), which is more than 30 A away from the reactive site, changed the inhibitor, Streptomyces subtilisin inhibitor (SSI), into a temporary inhibitor without a change in the inhibition constant. When the inhibitor was in excess of subtilisin BPN', the wild-type SSI was stable under protease attack, while the mutant inhibitor was hydrolyzed to peptide fragments in an all-or-none manner. The mechanism of this temporary inhibition induced by the amino acid substitution was studied on the basis of structural, thermodynamic, and kinetic data obtained by a combined use of NMR, hydrogen-deuterium exchange, differential scanning calorimetry, and gel filtration HPLC. The mutation did not induce major structural changes, and in particular, the structure of the enzyme-binding region was virtually unaffected. The denaturation temperature of SSI, however, was decreased by 10 deg upon mutation, although it still remained a thermostable protein with a denaturation temperature of 73 degrees C. Furthermore, the activation enthalpy for denaturation was reduced dramatically, to half that of the wild type. When the mutated SSI is present in excess of the enzyme, the proteolysis followed first-order reaction kinetics with respect to the total concentration of the mutated SSI molecules present. From these combined results, we conclude that the proteolysis proceeds not through the native form of the inhibitor in the inhibitor-enzyme complex but through the denatured (unfolded) form of the inhibitor whose fraction is increased by the mutation. This conclusion states that the necessary condition for being a serine protease inhibitor lies not only in the design of the reactive site structure that is highly resistant to protease attack but also in the suppression of such structural fluctuation that brings about cooperative denaturation. In contrast, when the protease existed in excess of the mutated inhibitor, the proteolysis reaction was accelerated by more than 2 orders of magnitude. Furthermore, the reaction occurred even in the wild-type SSI at a comparable rate as in the mutated protein. This indicates that in the enzyme excess case another, more efficient digestion mechanism involving fluctuation within the native manifold of the inhibitor dominates.     

应用定点突变与分子模建方法研究蛋白酶抑制剂eglin C突变体对蛋白酶furin和kexin的抑制活力

蛋白质前体加工酶参与许多重要蛋白质闪体的加工成熟过程,哺乳动物来源的furin和酵母中的kexin是该家族的重要成员。首先人工合成了编码枯草杆菌蛋白酶抑制剂eglin C的基因片段,组装后在大肠杆菌中得到表达。以定点突变方法在野生型eglin C抑制活性中心的P1、P2和P4位引入碱性氨基酸残基可以将其改造为很强的furin抑制剂（Ki约10^-9mol/L）,和kexin抑制剂（Ki约10^-11mol/L）。同时根据枯草杆菌蛋白酶和eglin C复合物的晶体结构,计算机同源模建了前体加工酶与eglin C突变体结构之间的相互作用,并结合实验数据得到以下结果：（1）P1位引入的碱性残基是该抑制剂活力的前提;（2）P4位碱性残基的引入可以极大地提高抑制剂活力约两个数量级;（3）P2 的碱性残基将有效提高抑制剂的活力。然而同时可以破坏抑制剂本身的稳定性。（4）野生型P3位的疏水性残基参与抑制剂活性环附近疏水核心的构成。     

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.