"Ars sine arte:" Nicholas Lemery and the end of alchemy in eighteenth-century France.
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Alchemy↗
Biomedical subjects
J C Powers
Publications and source records attributed to J C Powers.
Lower extremity purpura associated with new-onset autoimmune hepatitis: an unusual presentation.
Autoimmune hepatitis is a chronic necroinflammatory disorder of unknown cause. The morbidity and mortality of this potentially lethal disorder can be minimized by timely diagnosis and treatment. Patients can present with a wide variety of clinical and laboratory manifestations, including high titers of antinuclear antibody and other autoantibodies commonly associated with dermatologic disorders. Overt evidence of hepatic injury can be a late finding. An unusual presentation of autoimmune hepatitis and a brief review of its clinical features are presented, with an emphasis on those findings most relevant to a practicing dermatologist.
Adult↗
Reactions of [14C]-3,4-dichloroisocoumarin with subunits of pituitary and spleen multicatalytic proteinase complexes (proteasomes).
Exposure to [14C]-3,4-dichloroisocoumarin (DCI) of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary and spleen leads to label incorporation into several beta-type subunits, to rapid inactivation of the chymotrypsin-like (ChT-L) activity, and to a slower inactivation of other activities of the MPC. The pituitary and spleen MPCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde inhibitors of the ChT-L activity protected the X subunit in the pituitary MPC and unexpectedly the LMP2 subunit in the spleen MPC from label incorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit. Losses in the yield of amino acids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reaction of DCI with the threonine OH group. Brief exposure to [14C]-DCI led to preferential incorporation of label into the LMP2 and X subunits, consistent with the high inactivation rate constants of the ChT-L activity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO, an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating differences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spleen MPC, respectively, and suggest the catalytic functions of two other beta-subunits.
Animals↗
Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them.
The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.
Animals↗
Synthesis and kinetic studies of diphenyl 1-(N-peptidylamino)alkanephosphonate esters and their biotinylated derivatives as inhibitors of serine proteases and probes for lymphocyte granzymes.
Diphenyl 1-(N-peptidylamino)alkanephosphonate esters are highly reactive, specific, and aqueously stable irreversible inhibitors which can be used to probe the functions of many serine proteases, including the lymphocyte granzymes. We synthesized 16 peptide phosphonates with Ala, Met, Phe, or Val P1 amino acid residues, including two biotinylated derivatives for future functional and biochemical characterization of granzymes. The reactivity of the inhibitors was characterized with human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine chymotrypsin, and the granzymes of natural killer (NK) cells, which include a number of proteolytic activities (Asp-ase, Met-ase, etc.) that cleave peptide substrates with these residues in the P1 position. The reactivity and specificity of the phosphonates depended on the length and sequence of the peptidyl moiety and on the leaving group. Z-Ala-Ala-AlaP(OPh)2 was a good inhibitor of HLE and PPE (k(obsd)/[I] = 38 and 30 M(-1) s(-1), respectively) and had little reactivity with chymotrypsin. Z-Phe-Pro-Phe-P(OPh)2 was a good inhibitor of chymotrypsin (k(obsd)/[I] = 17,000 M(-1) s(-1)) and had little reactivity with the elastases. The leaving group of Z-MetP(OPh-4-Cl)2 made it a more effective chymotrypsin inhibitor than Z-MetP(OPh)2 (k(obsd)/[I] values of 142 and 30 M(-1) s(-1), respectively). With granzymes, the compounds reacted with a fraction of the Met-ase, chymase, and Ser-ase activities and lacked reactivity with Asp-ase and tryptase. Z-MetP(OPh-4-Cl)2 was an excellent inhibitor of Met-ase 1. Bi-Aca-Aca-Phe-Leu-PheP(OPh)2 appears to react specifically with one chymase while leaving other chymases untouched. Perforin-dependent lysis mediated by cytotoxic lymphocyte granules was inactivated by Z-Ala-Ala-AlaP(OPh)2, Z-MetP(OPh-4-Cl)2, Z-Leu-PheP(OPh)2, and Bi-Aca-Aca-Phe-Leu-PheP(OPh)2. The biochemical properties and biological efficacy of these inhibitors make them suitable for cellular and physiological studies of granzyme function.
Biotin↗
P-4 and RNKP-7, new granzyme-like serine proteases expressed in activated rat lymphocytes.
Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.
Amino Acid Sequence↗
The 1.8 A crystal structure of human cathepsin G in complex with Suc-Val-Pro-PheP-(OPh)2: a Janus-faced proteinase with two opposite specificities.
The crystal structure of human neutrophil cathepsin G, complexed with the peptidyl phosphonate inhibitor Suc-Val-Pro-PheP-(OPh)2, has been determined to a resolution of 1.8 A using Patterson search techniques. The cathepsin G structure shows the polypeptide fold characteristic of trypsin-like serine proteinases and is especially similar to rat mast cell proteinase II. Unique to cathepsin G, however, is the presence of Glu226 (chymotrypsinogen numbering), which is situated at the bottom of the S1 specificity pocket, dividing it into two compartments. For this reason, the benzyl side chain of the inhibitor PheP residue does not fully occupy the pocket but is, instead, located at its entrance. Its positively charged equatorial edge is involved in a favourable electrostatic interaction with the negatively charged carboxylate group of Glu226. Arrangement of this Glu226 carboxylate would also allow accommodation of a Lys side chain in this S1 pocket, in agreement with the recently observed cathepsin G preference for Lys and Phe at P1. The cathepsin G complex with the covalently bound phosphonate inhibitor mimics a tetrahedral substrate intermediate. A comparison of the Arg surface distributions of cathepsin G, leukocyte elastase and rat mast cell protease II shows no simple common recognition pattern for a mannose-6-phosphate receptor-independent targeting mechanism for sorting of these granular proteinases.
Amino Acid Sequence↗
Novel peptidyl alpha-keto amide inhibitors of calpains and other cysteine proteases.
A series of new dipeptidyl alpha-keto amides of the general structure R1-L-Leu-D,L-AA-CONH-R2 were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. They combine 10 different N-protecting groups (R1), 3 amino acids residues in P1 (AA), and 44 distinct substituents on the alpha-keto amide nitrogen (R2). In general, calpain II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors displaying dissociation constants (Ki) in the 10-100 nM range. Calpain I was also effectively inhibited, but very low Ki values were observed with a smaller number of inhibitors than with calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best inhibitors for calpain II were Z-Leu-Abu-CONH-CH2-CHOH-C6H5 (Ki = 15 nM), Z-Leu-Abu-CONH-CH2-2-pyridyl (Ki = 17 nM), and Z-Leu-Abu-CONH-CH2-C6H3(3,5(OMe)2) (Ki = 22 nM). The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH2-2-pyridyl (Ki = 19 nM). The peptide alpha-keto amide Z-Leu-Abu-CONH-(CH2)2-3-indolyl was the best inhibitor for cathepsin B (Ki = 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such inhibitors may be useful in elucidating the physiological and pathological events involving these proteases and may become possible therapeutic agents.
Animals↗
Inhibition of trypsin and thrombin by amino(4-amidinophenyl)methanephosphonate diphenyl ester derivatives: X-ray structures and molecular models.
X-ray structures of trypsin from bovine pancreas inactivated by diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl)methanephosphonate [Z-(4-AmPhGly)P(OPh)2] were determined at 113 and 293 K to 1.8 angstrom resolution and refined to R factors of 0.211 (113 K) and 0. 178 (293 K). The structures reveal a tetrahedral phosphorus covalently bonded to the O gamma of the active site serine. Covalent bond formation is accompanied by the loss of both phenoxy groups. The D-stereoisomer of Z-(4-AmPhGly)P-(OPh)2 is not observed in the complex. The L-stereoisomer of the inhibitor forms contacts with several residues in the trypsin active site. One of the phosphonate oxygens is inserted into the oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp194, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N epsilon 2 of His 57. The p-amidinophenylglycine moiety binds into the trypsin primary specificity pocket, interacting with Asp189. The amide forms a hydrogen bond to the carbonyl oxygen atom of Ser214. The inhibitor moiety, from the 113 K structure of trypsin inactivated by the reaction product of Z-(4-AmPhGly)P(OPh)2, was docked into human thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., & Hofsteenge, J. (1989) EMBO J. 8, 3467-3475] and energy minimized. The inhibitor fits well into the thrombin active site, forming favorable contacts similar to those in the trypsin complex with no bad contacts.
Animals↗
4-Chloro-7-(iodoacetyl)amino-3-methoxy-isocoumarin.
The crystal structure of the iodo analog of 7-(bromoacetyl)amino-4-chloro-3-methoxyisocoumarin, an inhibitor of human neutrophil elastase (HNE), C12H9ClINO4, has been determined. The isocoumarin ring system is highly planar, with the carbonyl group of the amide function being coplanar with the isocoumarin ring.
Coumarins↗
Determination of activable proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine protease inhibitor.
The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumarin serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIC). The presence of bound inhibitor on sperm was then determined by secondary labeling with avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expose the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples from six bulls, the mean proportion of sperm with activable proacrosin/acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed acrosin after cryopreservation of these samples was 55.8 +/- 4.1%. Significant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryopreservation. Activable proacrosin/acrosin levels in samples of cryopreserved sperm from five bulls were not correlated with fertility. These results do indicate, however, that the irreversible isocoumarin serine protease inhibitor BIC can be used to determine the proportion of sperm cells that retain activable proacrosin/acrosin after cryopreservation and thawing.
Acrosin↗
Novel pyridinium derivatives as inhibitors for acetylcholinesterase.
The carbamate 1-(methyl-3-(N,N-dimethylcarbamoyloxy)-2-pyridylmethylene)-4 -(4-phenyl) diazinecarboxamide chloride (MHP 133) is the parent for a new class of pyridinium salts which inhibit acetylcholinesterase (AChE) in vitro as well as in vivo. Fourteen new derivatives of MHP 133 have been synthesized with the intention of improving their hydrophobicity while maintaining their propensity to inhibit acetylcholinesterase. Upon prolonged incubation with AChE, the pyridinium salts exhibit progressive time-dependent inhibition according to first order kinetics with kobs/[I] values ranging from 3 to 345 M-1s-1. The enzyme did not regain any activity after prolonged incubation with the inhibitors (1 day). The partition coefficients for each inhibitor were evaluated in octanol/water in order to determine their hydrophobic character as hydrophobicity is a key prerequisite for crossing the blood brain barrier.
Acetylthiocholine↗
A kinetic study of the hydrolysis of the N-tosylalanine ester of 3-hydroxy-5-phenylpyrrole and related compounds by human leukocyte elastase.
The tosylalanine ester of 3-hydroxy-5-phenylpyrrole (HOPPy) is localized on reagent strips (Ames LEUKOSTIX) and used diagnostically to test urine for the presence of human leukocyte elastase (HLE) as an indication of urinary tract infection. We have determined the kinetic constants for the HLE-catalyzed hydrolysis of this substrate and the related substrates Tos-Ala-ONp, Cbz-Ala-OPPy, and Cbz-Ala-ONp, in solution at three different pH values. In the reagent strip matrix, diazo coupling of 4-diazo-3-hydroxy-1-napthylsulfonate (HONapN+2) with the enzymatic hydrolysis product HOPPy generates a purple color. We have also studied the kinetics of the reaction of HOPPy with HONapN+2 and other related diazonium salts such as 4-diazo-3-hydroxy-7-nitro-1-napthylsulfonate, 4-diazo-3-hydroxy-1,7-napthyldisulfonate, and 2-methoxy-4-(N-morpholinyl)benzene diazonium chloride. Tos-Ala-OPPy is the most reactive substrate among the compounds examined and kinetic studies indicate that deacylation is rate-limiting for HLE hydrolysis. The presence of decanol accelerates the enzymatic hydrolysis of Tos-Ala-OPPy with a kcat/KM = 10(7) M-1 s-1, which is close to the diffusion-controlled limit. For the diazo coupling reaction, the rate is affected by the substituents on the naphthalene ring and by the buffer in which the reaction occurs. This research has elucidated some important mechanistic features for the reaction of these compounds and may lead to improved methods for the detection of leukocyte elastase.
Alanine↗
X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design.
Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases involved in tissue remodeling. They have also been implicated in various disease processes including tumour invasion and joint destruction and are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is essential. Recently, we have published the refined high-resolution crystal structure of the catalytic domain of human neutrophil collagenase (HNC) complexed with the inhibitor Pro-Leu-Gly-NHOH, which is a mimic for the unprimed (P3-P1) residues of a bound peptide substrate. We have now determined two additional HNC complexes formed with the thiol inhibitor HSCH2CH(CH2Ph)CO-L-Ala-Gly-NH2 and another hydroxamate inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, which were both refined to R-values of 0.183/0.198 at 0.240/0.225-nm resolution. The inhibitor thiol and hydroxamate groups ligand the catalytic zinc, giving rise to a slightly distorted tetrahedral and trigonal-bipyramidal coordination sphere, respectively. The thiol inhibitor diastereomer with S-configuration at the P1' residue (corresponding to an L-amino acid analog) binds to HNC. Its peptidyl moiety mimics binding of primed (P1'-P3') residues of the substrate. In combination with our first structure a continuous hexapeptide corresponding to a peptide substrate productively bound to HNC was constructed and energy-minimized. Proteolytic cleavage of this Michaelis complex is probably general base-catalyzed as proposed for thermolysin, i.e. a glutamate assists nucleophilic attack of a water molecule. Although there are many structural and mechanistic similarities to thermolysin, substrate binding to MMPs differs due to the interactions beyond S1'-P1'. While thermolysin binds substrates with a kink at P1', substrates are bound in an extended conformation in the collagenases. This property explains the tolerance of thermolysin for D-amino acid residues at the P1' position, in contrast to the collagenases. The third inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, unexpectedly binds in a different manner than anticipated from its design and binding mode in thermolysin. Its hydroxamate group obviously interacts with the catalytic zinc in a favourable bidentate manner, but in contrast its isobutyl (iBu) side chain remains outside of the S1' pocket, presumably due to severe constraints imposed by the adjacent planar hydroxamate group. Instead, the C-terminal Ala-Gly-NH2 tail adopts a bent conformation and inserts into this S1' pocket, presumably in a non-optimized manner. Both the isobutyl side chain and the C-terminal peptide tail could be replaced by other, better fitting groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Mechanism-based isocoumarin inhibitors for human leukocyte elastase. Effect of the 7-amino substituent and 3-alkoxy group in 3-alkoxy-7-amino-4-chloroisocoumarins on inhibitory potency.
A series of 3-alkoxy-7-amino-4-chloroisocoumarins with various 3-alkoxy substituents have been prepared and evaluated as inhibitors of human leukocyte elastase (HLE). In addition, a new series of acyl, urea, and carbamate derivatives of 7-amino-4-chloro-3-methoxyisocoumarin (1), 7-amino-4-chloro-3-propoxyisocoumarin (3), and 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarin (6) have been synthesized. Most of the synthesized compounds are very potent inhibitors of HLE with kobs/[I] values between 10(4) and 10(6) M-1 s-1. Hydrophobic substituents on the 7-amino position of the isocoumarin ring afford the best selectivity and inhibitory potency for HLE. In the 2-bromoethoxy series, compound 24 with a PhNHCONH 7-substituent had a kobs/[I] value of 1.2 x 10(6) M-1 s-1, was very selective for HLE, and was the most potent inhibitor of HLE tested. Of the extended chain L-phenylalanyl derivatives, the Bz-L-Phe compound 66 with a kobs/[I] value of 1.8 x 10(5) M-1 s-1 was the most potent inhibitor of HLE in the 3-methoxyisocoumarin series and was also very selective for HLE. Our results indicate that a high degree of selectivity, along with potency, can be introduced into mechanism-based isocoumarin inhibitors.
Amino Acid Sequence↗
Mammalian tissue trypsin-like enzymes: substrate specificity and inhibitory potency of substituted isocoumarin mechanism-based inhibitors, benzamidine derivatives, and arginine fluoroalkyl ketone transition-state inhibitors.
Amino acid and peptide thioesters which contained Arg or Lys in the P1 position were tested as substrates for rat skin tryptase, and the kinetic constants Kcat/KM for the better substrates such as Z-Aba-Arg-SBzl, and Z-Gly-Arg-SBzl were over 5,000,000 M-1 s-1. The inhibitory potency of arginine fluoroalkyl ketones, benzamidine derivatives, and substituted isocoumarins containing basic functional groups was studied with rat skin tryptase, human lung tryptase, human skin tryptase, and bovine trypsin. 1-Naphthoyl-Arg-CF3 was the best arginine fluoroalkyl ketone reversible inhibitor for rat skin tryptase with a KI of 0.9 microM. 1-(4-Amidino-phenyl)-3-(4-phenoxyphenyl) urea showed competitive inhibition against bovine trypsin and rat skin tryptase with KI values of 2 and 4 microM, respectively. Isocoumarin derivatives with isothioureidoalkoxy substituents at the 3 position were potent irreversible inhibitors of these three tryptases with Kobs/[I] values of 10(4)-10(5) M-1 s-1. 4-Chloro-3-(2-isothioureido)ethoxy-7-phenylcarbamoylaminoisocou marin and 7-benzylcarbamoylamino-4-chloro-3-(3-isothioureido)propox yisocoumarin inactivated trypsin and formed stable trypsin-inhibitor complexes which regained less than 8% of activity upon standing in the pH 7.5 buffer and regained 30-75% of activity in the presence of 0.3 M NH2OH after 1 day. In contrast, the complexes with rat skin tryptase regained activity rapidly, indicating differences in the inhibition mechanism and active site structures of these related enzymes.
Amino Acid Sequence↗
Peptide thioester substrates for serine peptidases and metalloendopeptidases.
Peptide thioesters are sensitive substrates of various serine peptidases and metalloendopeptidases. Thioester substrates generally have high enzymatic hydrolysis rates and low background hydrolysis rates, and the hydrolysis rates can be easily monitored in the presence of thiol reagents such as 4,4'-dithiodipyridine or 5,5'-dithiobis (2-nitrobenzoic acid). Peptide thioester substrates have been invaluable for the study of enzyme specificity and enzyme inhibitors, especially in cases where no other practical synthetic substrates are available. Tripeptide substrates of the type Boc-Ala-Ala-AA-SBzl, where AA is nearly all of the 20 common amino acids, have now been synthesized and should be useful for the subsite mapping of new serine peptidases and the study of crude cell preparations containing serine peptidases.
Amino Acid Sequence↗
Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylating agents.
Acylated derivatives of thrombin have been made using low molecular weight acylating agents, carbamylating agents and carbonylating agents. The compounds used to acylate the active site serine include isatoic anhydrides, benzoxazinones, benzylisocyanate, N-(benzylcarbonyloxy)succinimide and p-(dimethylamino)benzoylimidazolide. The rates of acylation and deacylation were determined. The best overall inhibitors of thrombin are 2-ethoxy-4H-3,1-benzoxazin-4-one, isatoic anhydride and tert-butyl-2,4-dioxo-2H-3,1-benzoxazine-1(4H)-acetate, which have k2/Ki values of 52,700 M-1s-1, 48,900 M-1s-1 and 5400 M-1s-1, respectively. The carbamyl derivative of thrombin formed with benzylisocyanate had the slowest rate of deacylation (2.3 x 10(-7) s-1), while the ester derivative formed with 2-(N,N-dimethylamino)methylimino-4H-3,1-benzoxazin-4-one had the fastest rate of deacylation (1.9 x 10(-4) s-1).
Acylation↗