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PubMed1999

Modification of the thermoresistance to spray-drying of a cold-adapted subtilisin by genetic engineering.

The thermoresistance of a cold-adapted subtilisin dried by spray-drying was studied. Proteolytic activity of this enzyme was measured before and after spray-drying. Without chemical additives, spray-drying yields ranged from 2-13%. The use of arabic gum and lactose in the composition of the enzyme solutions allowed the strengthening of the enzyme structures and increased water mobility in the product. Increase of water mobility led to a shorter residence time of the product in the spray-drier and a net yield increase was obtained (yield higher than 50%). The effect of two selective mutations on the thermoresistance to spray-drying of the cold-adapted subtilisin was also investigated. Mutation T85D (introduction of an additional link with an ion Ca2+ necessary for enzyme activity, by substitution of Asp for Thr 85) had no effect on the thermoresistance of the subtilisin to spray-drying. Mutation H121W (introduction of an additional aromatic link by substitution of Trp for His 121) reduced the drying yield from 66% (not modified subtilisin) to 52%. This higher thermosensitivity could be explained by an increase of the hygroscopic character of the modified subtilisin (mutation H121W).

Journal Article↗
PubMed2000

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.

Acetonitriles↗
PubMed1987

Polymorphic assembly of subtilisin-cleaved tubulin.

Limited proteolysis of tubulin with subtilisin results in cleavage of both the alpha and beta subunits, releasing small peptides from the C-terminal ends. At 37 degrees C the digested tubulin assembles into polymorphic structures: microtubules with attached ribbons in the presence of GTP, rings in the presence of GDP, and protofilament spirals in the presence of vinblastine. Undigested tubulin does not assemble under these conditions. Rings and Vinca-induced spiral structures are assembled from undigested tubulin only when microtubule-associated proteins, high Mg2+ concentrations, or polycations are present. Thus, cleavage with subtilisin affects assembly in a manner similar to the addition of these agents. It appears that binding of positively charged substances may act by neutralizing the charge on the highly acidic C-terminal regions of the alpha- and beta-subunits, while cleavage with subtilisin produces the same effect by removing these peptides. Undigested and subtilisin-digested tubulin form sheets of protofilaments in the presence of Zn2+, which indicates that the binding sites for the 2-3 Zn2+ ions necessary to induce sheet formation do not reside in the C-terminal regions of the monomers.

Animals↗
PubMed1996

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state.

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10)s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8)s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8)s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10)s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.

Bacterial Proteins↗
PubMed1989

Thermitase, a thermostable subtilisin: comparison of predicted and experimental structures and the molecular cause of thermostability.

The subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three-dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserved structural core and less well in seven loop regions involving insertions and deletions relative to subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular cause of thermostability we confront hypotheses derived by calculation of the details of interatomic interactions and estimates of hydrophobic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main causes of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.

Actinomycetales↗
PubMed1989

Engineering subtilisin BPN' for site-specific proteolysis.

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.

Amino Acid Sequence↗
PubMed1997

Hydrophobic ion pairing as a method for enhancing structure and activity of lyophilized subtilisin BPN' suspended in isooctane.

The use of enzymes in low water environments permits reactions to occur that are difficult or impossible in aqueous solution. In this manner, proteases can be used to form, rather than hydrolyze, ester and amide linkages. Presumably, the native-like structure of the enzyme must remain intact for catalysis to transpire. However, little is known regarding the integrity of the overall structure of lyophilized proteins suspended in organic media. In this study, the structural changes that occur during the freeze-drying process and those effected by suspension in the organic solvent were examined. Using Fourier-transform infrared spectroscopy, the secondary structure of lyophilized subtilisin BPN' was monitored and correlated to the level of enzymatic activity when suspended in isooctane. In addition, the ability of ionic detergents to stabilize subtilisin BPN' via ion pairing was evaluated. It was found that subtilisin unfolds to some degree during lyophilization, whether it is ion paired or not. Furthermore, there are structural changes observed when the enzyme is placed in isooctane, although the effects are less with ion-paired subtilisin. This higher level of retention of secondary structure results in increased enzymatic activity.

Detergents↗
PubMed1996

Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis.

Alkaline elastase YaB, produced by alkalophilic Bacillus YaB, is an extracellular serine protease having 55% homology to subtilisin BPN' and thus could be called subtilisin YaB. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine if the pro-peptide of subtilisin YaB functions in trans to guide the folding of secreted subtilisin YaB in vivo, we made genes encoding the pre-pro, pro and pre-mature portions and placed them under the control of the spac-1 promoter on a multi-copy plasmid. When simultaneous expression in Bacillus subtilis of both the pre-pro and pre-mature genes was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG), protease activity was detected in the medium. On the other hand, we could not detect protease activity when the expression of either the pre-mature gene alone or both the pro and pre-mature genes was induced. From these results, we concluded that the pro region functions in trans and outside the cells for the proper folding and activation of the enzyme.

Bacillus subtilis↗
PubMed1993

Requirement for a disulfide bridge near the reactive site of protease inhibitor SSI (Streptomyces subtilisin inhibitor) for its inhibitory action.

The problem of why serine protease inhibitors having substrate-like structure around the reactive site are not degraded by the cognate protease has prompted us to investigate the structural requirements in Streptomyces subtilisin inhibitor (SSI) for its inhibitory action. We removed the disulfide bridge between Cys71 and Cys101 near the reactive site by oligonucleotide-directed mutagenesis, replacing both Cys residues with Ser residues. Inhibitory activity of the mutated SSI toward subtilisin BPN' was initially potent, but decreased remarkably with increasing incubation time after mixing (temporary inhibition), due to degradation of the mutated SSI by subtilisin via a specific intermediate with a nick at the reactive site (Met73-Val74). Binding affinity of subtilisin for the mutated SSI was reduced by more than 10(3)-fold, and the mutated SSI showed a 20 deg.C decrease in melting temperature, which probably mainly reflects the destruction of the region of alpha-helix containing Cys101. These results imply that the susceptibility of the mutated SSI to protease, and the irreversibility of the peptide bond cleavage at the reactive site, result from increased flexibility around the reactive site in the complex of the disulfide-bond-removed SSI with the protease, demonstrating the requirement of conformational rigidity around the reactive site of the inhibitor for its native inhibitory action.

Amino Acid Sequence↗
PubMed1999

Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins.

High-alkaline serine proteases have been successfully applied as protein degrading components of detergent formulations and are subject to extensive protein engineering efforts to improve their stability and performance. Dynamics has been suggested to play an important role in determining enzyme activity and specificity and it is therefore of interest to establish how local changes in internal mobility affect protein stability, specificity and performance. Here we present the dynamic properties of the 269 residue serine proteases subtilisin PB92 (Maxacal(TM)) and subtilisin BLS (Savinase(TM)), secreted by Bacillus lentus, and an engineered quadruple variant, DSAI, that has improved washing performance. T1, T2 and heteronuclear NOE measurements of the 15N nuclei indicate that for all three proteins the majority of the backbone is very rigid, with only a limited number of residues being involved in local mobility. Many of the residues that constitute the S1 and S4 pockets, determining substrate specificity, are flexible in solution. In contrast, the backbone amides of the residues that constitute the catalytic triad do not exhibit any motion. Subtilisins PB92, BLS and DSAI demonstrate similar but not identical NMR relaxation rates. A detailed analysis of local flexibility indicates that the motion of residues Thr143 and Ala194 becomes more restricted in subtilisin BLS and DSAI. Noteworthy, the loop regions involved in substrate binding become more structured in the engineered variant as compared with the two native proteases, suggesting a relation between altered dynamics and performance. Similar conclusions have been established by X-ray crystallograpic methods, as shown in the accompanying paper.

Bacillus↗
PubMed1994

Improvement of a useful enzyme (subtilisin BPN') by an experimental evolution system.

In order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of successive in vitro random mutagenesis and efficient screening systems. Subtilisin BPN', a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector system. Primary mutants with reduced activities of below 80% of that of the wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone non-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detecting transformant colonies incubated at 10 degrees C with clear zones that were greater in size than that of the wild type. One such secondary mutant, 12-12, derived from a primary mutant with 80% activity, was found to gain 150% activity (kcat/Km value) of the wild-type when the mutant subtilisin gene was subcloned to a Bacillus subtilis host-vector system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate. When N-succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide was used, 180% activity was gained. Genetic analysis revealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca(2+)-binding ability. The thermostability was also found to be related to the activity.

Amino Acid Sequence↗
PubMed1999

Chemical engineering of enzymes: altered catalytic activity, predictable selectivity and exceptional stability of the semisynthetic peroxidase seleno-subtilisin.

The increasing demand for enzymes as highly selective, mild, and environmentally benign catalysts is often limited by the lack of an enzyme with the desired catalytic activity or substrate selectivity and by their instability in biotechnological processes. The previous answers to these problems comprised genetically engineered enzymes and several classes of enzyme mimics. Here we describe the potential of chemical enzyme engineering: native enzymes can be modified by merely chemical means and basic equipment yielding so-called semisynthetic enzymes. Thus, the high substrate selectivity of the enzymatic peptide framework is combined with the catalytic versatility of a synthetic active site. We illustrate the potential of chemically engineered enzymes with the conception of the semisynthetic peroxidase seleno-subtilisin. First, the serine endoprotease subtilisin was crystallized and cross-linked with glutaraldehyde to give cross-linked enzyme crystals which were found to be insoluble in water or organic solvents and highly stable. Second, serine 221 in the active site (Enz-OH) was chemically converted into an oxidized derivative of selenocystein (Enz-SeO2H). As a consequence, the former proteolytic enzyme gained peroxidase activity and catalyzed the selective reduction of hydroperoxides. Due to the identical binding sites of the semisynthetic peroxidase and the protease, the substrate selectivity of seleno-subtilisin was predictable in view of the well-known selectivity of subtilisin.

Binding Sites↗
PubMed2005

Phylogenetic and exon-intron structure analysis of fungal subtilisins: support for a mixed model of intron evolution.

Phylogenetic and exon-intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon-intron structure that the homogeneous sequences usually have a similar exon-infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.

Amino Acid Sequence↗
PubMed2003

Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate.

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.

Alkanesulfonic Acids↗
PubMed1985

Structural changes of rabbit liver fructose-1,6-bisphosphatase: the effect of urea and subtilisin digestion.

At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P2-ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that the pK value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP (I 0.5), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding.

Adenosine Monophosphate↗
PubMed1991

Studies of crystallization conditions for native and subtilisin-cleaved pig brain tubulin.

A survey of crystallization conditions for pig brain tubulin, using standard vapor diffusion techniques in sitting drops or capillaries, has resulted in irregular, fragile needles or plates with a largest dimension of 0.5 mm. These occurred in 2.5% PEG (MW 3350), 0.1 M Pipes, pH 6.2 and 6.4, 2-16 mM MgSO4, 10-15 mM DTE, and 0.1 mM GDP at 8 degrees C. When GTP replaced GDP these aggregates did not form under any of the conditions surveyed (temperature: 8-10 degrees C; MgSO4: 2-16 mM; pH 6-7; PEG, MW 3350: 1.25-12.5%). EM observations demonstrated that sheets of rings appear in crystal solutions in the presence of GDP or GTP. These results are consistent with the results of Howard and Timasheff (1986, Biochemistry 25, 8292-8300) that tubulin rings form in the presence of GDP or GTP but more readily in GDP. Tubulin crystallization experiments are hampered by tubulin's high degree of heterogeneity. Much of the variability lies in the carboxyl terminal region. Conditions for limited digestion of the heterodimer by subtilisin, removing only the carboxyl terminus, were determined. Reduction of heterogeneity was demonstrated by isoelectric focusing. The solubilities of native and subtilisin-cleaved tubulin in MgSO4, (NH4)2SO4, PEG (MW 1450, 3350, 10,000), DMSO, and MPD were compared. Subtilisin-cleaved tubulin precipitated more readily than native tubulin under all conditions surveyed, consistent with the removal of the highly acidic carboxyl terminus. Vapor diffusion experiments using subtilisin-cleaved tubulin under conditions where native tubulin forms needles or plates resulted in similar aggregates.

Animals↗
PubMed1976

Denaturation of subtilisin BPN' and its derivatives in aqueous guanidine hydrochloride solutions.

The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible.

Guanidines↗
PubMed1977

Inhibition of subtilisin BPN' with peptide chloromethyl ketones.

The kinetics of the reaction of a series of peptide chloromethyl ketones with subtilisin BPN' (EC 3.4.21.14) were investigated in order to correlate the rates of reaction in solution with the number of interactions which are observed in the cystallographic model of the inhibited enzyme (Robertus, J.D., Alden, R.A., Birktoft, J.J., Powers, J.C. and Wilcox, P.E. (1972) Biochemistry 11, 2439--2449). The second-order rate constant kobs/[I], measured at pH 7.0 in 9% 1,2-dimethoxyethane at 30 degress C, varied by a factor of 5900 from the slowest (Z-TrpCH2Cl) to the fastest inhibitor (Ac-Phe-Gly-Ala-LeuCH2Cl). Inhibitors with a P1 leucine or phenylalanine residue are equally effective. The increased reactivity of inhibitors containing alanine as the P2 residue is the result of a favorable contact between the methyl side chain of the alanyl residue and the S2 subsite of subtilisin. This result correlates nicely with the previously observed "secondary specificity" of subtilisin for substrates with alanine as the P2 residue. Teetrapeptide and tripeptide chloromethyl ketone inhibitors have kobs/[I] values of over 100-fold greater than those of most dipeptide and amino acid chloromethyl ketones. This again agress with the crystallographic model since tripeptide and tetrapeptide inhibitors could form a beta-sheet structure involving three or four hydrogen bonds with the enzyme while the others would form fewer hydrogen bonds. The S4 subsite of subtilisin exhibits a distinct preference for aromatic groups and our four most reactive inhibitors (Ac-Phe-Gly-Aca-LeuCH2Cl, Z-Gly-PheCH2Cl, Z-Gly-Gly-LeuCH2Cl, and Boc-Ala-Gly-PheCH2Cl) have either an aromatic (or a large hydrophobic) group as the P4 residue. The results demonstrate that the solution reactivity of peptide chloromethyl ketones can be explained on the basis of the crystal structures of chloromethyl ketone-inhibited substilisin derivatives and that substrate hydrolysis rates can be used to design effective chloromethyl ketone inhibitors for serine proteases.

Amino Acid Chloromethyl Ketones↗