The mechanism of replication of phi x174 DNA. XVI. Evidence that the phi x174 viral strand is synthesized discontinuously

Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l-phenylalaninal residue of the inhibitor and the O^γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 Å difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l-phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.     

Mapping of multiple phosphorylation sites within the structural and catalytic domains of the Fujinami avian sarcoma virus transforming protein.

The phosphorylation sites of the P140gag-fps gene product of Fujinami avian sarcoma virus have been identified and localized to different regions of this transforming protein. FSV P140gag-fps isolated from transformed cells is phosphorylated on at least three distinct tyrosine residues and one serine residue, in addition to minor phosphorylation sites shared with Pr76gag. Partial proteolysis with virion protease p15 or with Staphylococcus aureus V8 protease has been used to generate defined peptide fragments of P140gag-fps and thus to map its phosphorylation sites. The amino-terminal gag-encoded region of P140gag-fps contains a phosphotyrosine residue in addition to normal gag phosphorylation sites. The two major phosphotyrosine residues and the major phosphorserine residue are located in the carboxy-terminal portion of the fps-encoded region of P140gag-fps. P140gag-fps radiolabeled in vitro in an immune complex kinase reaction is phosphorylated at only one of the two C-terminal tyrosine residues phosphorylated in vivo and weakly phosphorylated at the gag-encoded tyrosine and at a tyrosine site not detectably phosphorylated in vivo. Thus, the in vitro tyrosine phosphorylation of P140gag-fps is distinct from that seen in the transformed cell. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three Fujinami avian sarcoma virus variants showed that the phosphotyrosine-containing peptides are invariant, and this high degree of sequence conservation suggests that these sites are functionally important or lie within important regions. The P105gag-fps transforming protein of PRCII avian sarcoma virus lacks one of the C-terminal phosphotyrosine sites found in Fujinami avian sarcoma virus P140gag-fps. Partial trypsin cleavage of FSV P140gag-fps immunoprecipitated with anti-gag serum releases C-terminal fragments of 45K and 29K from the immune complex that retain an associated tyrosine-specific protein kinase activity. This observation, and the localization of the major P140gag-fps phosphorylation sites to the C-terminal fps region, indicate that the kinase domain of P140gag-fps is located at its C terminus. The phosphorylation of P140gag-fps itself is complex, suggesting that it may itself interact with several protein kinases in the transformed cell.     

An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides

Seeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (alphaNeuAc(2-3)betaGal(1-4)betaGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (alphaNeuAc(2-3)betaGal(1-3)[alphaNeuAc(2-6)]alphaG alNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-A resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensis hemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations.     

Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1', glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.     

Inflorescence bud protease(s) against dehydrin-like inflorescence bud proteins of Pistacia vera

This paper reports on a proteolytic activity extracted from Pistacia vera L. (pistachio) inflorescence buds, found in male and female tissues. The proteolytic activity reaches a peak at the beginning of bud opening and flowering and is directed against inflorescence bud dehydrin-like proteins of pistachio, IBP32 and IBP27. The effect of protease inhibitors indicated that the protease(s) belong to a serine-protease-like family and are not cysteine, acid or metallo-proteases. The proteolytic activity was strongly inhibited by Nα-p-tosyl-L-lysine chloromethyl ketone and to a lesser extent by N-tosyl-L-phenylalanine chloromethyl ketone, suggesting mainly trypsin-like specificity or broader serine-protease specificity. It is suggested that the proteolytic activity is important in the mobilization of nitrogen reserves stored in the bud storage proteins during dormancy to support the fast-developing inflorescence in spring after bud dormancy break.     

Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr(547) and a water molecule positioned in close proximity to Tyr(547). To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.     

Role of tryptophan hydroxylase phe313 in determining substrate specificity

The active site residue phenylalanine 313 is conserved in the sequences of all known tryptophan hydroxylases. The tryptophan hydroxylase F313W mutant protein no longer shows a preference for tryptophan over phenylalanine as a substrate, consistent with a role of this residue in substrate specificity. A tryptophan residue occupies the homologous position in tyrosine hydroxylase. The tyrosine hydroxylase W372F mutant enzyme does not show an increased preference for tryptophan over tyrosine or phenylalanine, so that this residue cannot be considered the dominant factor in substrate specificity in this family of enzymes.     

Studies on Bacterial Protease

Substrate specificity of the crystalline neutral protease of B. amylosacchariticus was investigated using the B-chain of oxidized beef insulin as the substrate, and the results were compared with those of proteases obtained from other strains of Bacillus subtilis. The neutral protease split the B-chain at eleven sites of the peptide linkages, indicating the narrow specificity as compared with subtilopeptidase A, The results also indicated that the peptide bonds susceptible to the action of the neutral protease were mainly those involving amino group of hydrophobic amino acids and tyrosine, with a few exception. The enzyme showed potent activities in casein digestion at near neutrality and in milk clotting at pH 5.6, whereas it was completely inert on esters and keratin, and only slightly active toward elastin.     

ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.     

Prediction of substrate specificity in NS3/4A serine protease by biased sequence search threading

Proteases recognize specific substrate sequences and catalyze the hydrolysis of targeted peptide bonds to activate or degrade them. It is particularly important to identify the recognition and binding mechanisms of protease–substrate complex structures in studies of drug development. Cleavage specificity in protease systems is generally determined by the amino acid profile, structural features, and distinct molecular interactions. In this work, substrate variability and substrate specificity of the NS3/4A serine protease encoded by the hepatitis C virus (HCV) was investigated by the biased sequence search threading (BSST) methodology. The available crystal structures of peptide-bound protease were used as templates as well as new complex structures that were generated via docking calculations. Threading various binding and nonbinding sequences as starting sequences over multiple templates, the potential sequence space was efficiently explored by a low-resolution knowledge-based scoring potential. The low-energy substrate sequences generated by the biased search are correlated with the natural substrates with conserved amino acid preferences, although some positions exhibit variability. Specifically, the amino acids which play essential roles in cleavage are mostly preferred. Potential substrate sequences were predicted by statistical probability approaches that consider the pairwise and triplewise interdependencies among residue positions in the low-energy sequences. The predicted substrate sequences also reproduce most of the natural substrate sequences, implying the complex interdependence between the different substrate residues. Consequently, the BSST seems to provide a powerful methodology for predicting the substrate specificity for the NS3/4A protease, which is a target in drug discovery studies for HCV.     

Model of a specific interaction: Salt-bridges form between prothrombin and its activating enzyme blood clotting factor Xa

In order for intricate biochemical pathways to function properly in the heterogeneous environment of a cell or organism, high specificity of enzymesubstrate reactions is essential. The molecular nature of this specificity is examined for the case of the specific activation of prothrombin by factor X_a of the blood coagulation system using model-building methods.The structure of blood clotting factor X_a was modeled by homology to the known structures of the mammalian serine proteases. The sequence about the cleaved peptide bond in prothrombin was placed into the active site of factor X_a in a conformation similar to that of bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor, when bound to trypsin, and to that of the tripeptide chloromethyl ketones, when bound to γ-chymotrypsin and Streptomyces griseus protease B.The model of the complex between prothrombin and factor X_a shows the expected salt-bridge between the Arg preceding the cleaved peptide bond and the primary specificity residue Asp189 of factor X_a. Two other salt-bridges appear to be formed: between a Glu three residues before the cleaved bond in prothrombin and Arg143 of factor X_a, and between a second Glu three residues after the cleaved bond and Lys62 in factor X_a. Several hydrogen bonds and hydrophobic interactions also occur in the complex. A stereogeometric requirement for a Gly two residues before the cleaved bond is also imposed by the factor X_a structure.Examination of the known serine protease sequences and model building suggest that the two new salt-bridges are unique to the prothrombin-factor X_a complex. Therefore, these charge interactions, and the requirement for a Gly, are likely to be at least partially responsible for the high specificity of the activation of prothrombin by factor X_a.     

A serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells

We report here by using stopped-flow fluorometry with three different fluorescent probes that a serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells. When cytotoxic T cells (mouse LC7, H-2b anti H-2d) bound to the specific target cells (mouse mastocytoma P815, H-2d), cytotoxic T cells first increased their membrane fluidity, and calcium then was released from intracellular stores. After that, there was a calcium influx from the external medium into the T cells. All of these steps, however, were blocked by serine protease inhibitors (soybean trypsin inhibitor, N alpha-p-tosyl-L-lysine chloromethyl ketone and tosylphenylalanyl chloromethyl ketone). Bovine pancreatic trypsin and chymotrypsin in the external medium mimicked the signalling events which were triggered by the serine protease on the T cell surfaces. From the reaction time (within 1 s) and its specificity, this serine protease in cytotoxic T cells was considered to be different from a protease which works at the killing stage.     

Double-headed protease inhibitors from black-eyed peas. II. Structural studies by optical absorption and circular dichroism.

Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 83 amino acid residues per monomer. Black-eyed pea trypsin inhibitor (BEPTI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 75 residues per monomer. The molar extinctions at 280 nm are 2770 for BEPCI and 3440 for BEPTI. The single tyrosyl residue is very inaccessible to solvent in native BEPCI and BEPTI at neutral pH and titrates anomalously with an apparent pK = 12. Ionization of tyrosine is complete in 13 hours above pH 12. No heterogeneity of the local environment of the tyrosyl residues in different subunits can be detected spectrophotometrically. The large number of cystine residues leads to an intense and complex near-ultraviolet CD spectrum with cystine contributions in the regions of 248 and 280 nm and tyrosine contributions at 233 and 280 nm. An intact disulfide structure is required for appearance of the tyrosyl CD bands. The inhibitors are unusually resistant to denaturation when compared with similar low molecular weight proteins of high disulfide content. All observations are consistent with a far more rigid structure for BEPCI and BEPTI than for a typical protein.     

Structural and electronic properties of the liver fluke heme cavity by nuclear magnetic resonance and optical spectroscopy. Evidence for a distal tyrosine residue in a normally functioning hemoglobin

Structural features of the heme and the heme cavity of the monomeric hemoglobin (Hb) from the platyhelminth Dicrocoelium dendriticum were investigated by optical and proton nuclear magnetic resonance spectroscopy. Using nuclear Overhauser effects (NOEs) from resonances assigned previously through isotope labeling, most hyperfine-shifted resonances could be attributed to individual heme and protein protons in the cyano-metHb complex. It was observed that the heme 2-vinyl group is held in the trans orientation by nearby residues, whereas the 4-vinyl group exhibits an equilibrium between cis and trans orientations. NOE experiments in 1H2O allowed the identification of exchangeable protons belonging to the proximal histidine residue (F8) and to a distal residue. Detailed analysis of the NOE patterns obtained from the distal labile proton to non-labile protons and among these latter protons leads to the conclusion that a tyrosine side-chain occupies the distal site E7. Optical spectra of the alkaline-metHb also lead to this view, in that they are not typical of a hydroxy-metHb complex but instead resemble that of a hemin-phenolate or human mutant (M-type) Hb with a tyrosine residue linked to the iron atom. Further evidence for a distal tyrosine residue stems from the occurrence of an unusually stable transient ferrous Hb-cyanide complex, formed upon reduction of cyano-metHb to deoxy-Hb with dithionite. We suggest that the stability of this intermediate is due to a slow re-orientation of a large distal side-chain prior to cyanide dissociation. The sequence of the E-helix, known from the partially determined primary structure, was realigned to accommodate these findings. A frame-shift by one residue now positions a tyrosine at the distal site E7 instead of the originally proposed glycine residue.     

Inhibition of neutrophil sulfhydryl groups by choloromethyl ketones. A mechanism for their inhibition of superoxide production

The inhibition of O2- production by serine protease inhibitors such as chloromethyl ketone derivatives, has been used as evidence to indicate that protease activity is essential for the production of O2- by neutrophils. However, chloromethyl ketones are potent inhibitors of sulfhydryl groups. This study demonstrates that chloromethyl ketones inhibited non-protein sulfhydryl groups as well as O2- production by human neutrophils stimulated with phorbol myristate acetate (PMA). Their inhibition of O2- production could be prevented by reduced glutathione. The results suggest that inhibition of O2- production by chloromethyl ketones is largely due to their inhibition of sulfhydryl groups.     

Preparation of monoclonal antibodies to phosphotyrosine and their use for identification of phosphotyrosine-containing proteins

Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates.     

Critical ionizing groups in Aeromonas neutral protease

Aeromonas neutral protease possesses two residues critical to its activity. One has a pKa of 5.5 in both the free enzyme and the enzyme-substrate complex and must be deprotonated for maximal activity. The other, which ionizes at pH 7.1 in the free enzyme and at pH 7.4 in the enzyme-substrate complex, must be protonated for optimal enzyme action. The protease is reversibly inhibited by aminoacyl hydroxamates, peptides containing a phenylalanyl residue, phosphoryl-L-phenylalanylglycylglycine, and by beta-phenylpropionyl-L-phenylalanine. The pH dependence of inhibition by the latter revealed that a residue with a pKa of 5.6 influences inhibitor binding. Compounds containing both a hydroxamido group and a chloroacetyl group are particularly effective in inactivating the enzyme, and inhibition is enhanced by hydrophobic residues. Thus, a 33-fold molar excess of chloroacetyl-N-hydroxy-L-phenylalanyl-L-alanyl-L-alanine amide rapidly inactivated Aeromonas neutral protease. Carbethoxylation experiments resulted in a 90% loss in activity which was fully reversible by hydroxylamine; spectral analysis indicated the involvement of a single histidine residue. Protection against both esterification and carbethoxylation was furnished by the presence of beta-phenylproprionyl-L-phenylalanine. Inactivation experiments suggest that a glutamic or aspartic acid and a histidine residue are responsible for the pKa values revealed by pH dependence studies.     

Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.     

Conversion of trypsin to a functional threonine protease

The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10(2) -10(4) -fold less active than wild type in kcat/KM but >10(6)-fold more active than the Ser 195 --> Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'- P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195.