Evidence for the general base mechanism in carboxypeptidase A-catalyzed reactions: partitioning studies on nucleophiles and H2(18)O kinetic isotope effects.

Methanol does not detectably compete with water in carboxypeptidase-catalyzed cleavage of any substrate, although it is preferentially reactive in a model for the proposed nucleophilic mechanism for the enzyme that involves an anhydride intermediate. To test for such a common intermediate in the cleavage of related peptide and ester substrates, a method has been developed to examine H2(16)O-H2(18)O kinetic isotope-partitioning effects. The finding that benzoylglycylphenylalanine has an isotope effect of 1.019 +/- 0.002 while benzoylglycyl-beta-L-phenyl-lactate shows a small inverse isotope effect excludes most versions of a nucleophilic mechanism having a common anhydride intermediate. The bulk of the available evidence strongly favors the previously proposed general base mechanism.     

Mechanism of action of carboxypeptidase A in ester hydrolysis.

The reaction of carboxypeptidase A (peptidyl-L-amino-acid hydrolase; EC 3.4.12.2) with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate has been investigated in the temperature range 25 degrees to -40 degrees with use of organic-aqueous cosolvent mixtures. In the subzero temperature range the hydrolysis reaction is characterized by a biphasic decrease in absorbance specific for the substrate. The kinetic data can be unambigously analyzed as two consecutive first-order reactions with formation of a covalent acyl-enzyme intermediate. Deacylation of the covalent intermediate is shown to be rate-limiting in the subzero temperature range, and near -60 degrees it is sufficiently stable for spectral characterization. Consideration of the structure of the active site and of the catalytically functional residues of the enzyme leads to the conclusion that the intermediate is a mixed anhydride in which the gamma-carboxylate of glutamate-270 is acylated by the substrate. The temperature dependence of the rate constants of the acylation and deacylation steps explains why the intermediate of this enzyme-catalyzed reaction is observed only at low temperatures.     

Zinc environment and cis peptide bonds in carboxypeptidase A at 1.75-A resolution.

The structure of the metalloenzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) has been refined at 1.75 A by a restrained least-squares procedure to a conventional crystallographic R factor of 0.162. Significant results of the refined structure relative to the catalytic mechanism are described. In the native enzyme, the zinc coordination number is five (two imidazole N delta 1 nitrogens, the two carboxylate oxygens of glutamate-72, and a water molecule). In the complex (at 2.0-A resolution) of carboxypeptidase A with the dipeptide glycyl-L-tyrosine, however, the water ligand is replaced by both the carbonyl oxygen and the amino nitrogen of the dipeptide. The amino nitrogen also statistically occupies a second position near glutamate-270. Consequently, the coordination number of zinc may vary from five to six in carboxypeptidase A-substrate complexes. Implications of these results for the catalytic mechanism of carboxypeptidase A are discussed. In addition, three cis peptide bonds, none of which involves proline as the amino nitrogen donor, have been located fairly near the active site.     

Cryospectrokinetic characterization of intermediates in biochemical reactions: carboxypeptidase A.

Cryospectrokinetic studies provide concurrent structural, kinetic, and chemical data on short-lived intermediates in the course of the interactions of enzymes with their substrates and of other, similar pairs of biomolecules. Subzero temperatures extend the lifetimes of these intermediates and, combined with rapid-mixing and rapid-scanning instrumentation, allow simultaneous measurement of both their physical-chemical and kinetic characteristics. For carboxypeptidase A, the spectra of a chromophoric, enzymatically functional cobalt atom at the active site signal the structure of the coordination complex during catalysis, while radiationless energy transfer between enzyme tryptophans and the fluorescent dansyl blocking group of rapidly hydrolyzed peptide and ester substrates provides the basis for measurement of the rates of formation and breakdown of intermediates. Subzero radiationless energy transfer kinetic studies of the zinc and cobalt enzymes disclose two intermediates in the hydrolysis of both peptides and esters and furnish all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2----E + P. The chemical and kinetic data indicate that neither of these is an acylenzyme intermediate. Both absorption and EPR spectra of the ES2 reaction intermediates consistently demonstrate the formation of transient metal complexes, differences between the effects induced by peptides and esters, and strong similarities between those induced by all peptides on the one hand and all esters on the other. The marked alterations of the cobalt spectra likely reflect the coordination of a substrate carboxyl and/or carbonyl group to the metal at a critical step in the course of catalysis. The cryospectrokinetic approach developed here in the mechanistic study of this metalloenzyme is applicable to the examination of transients of biochemical reactions in general. It will allow molecular characterization of previously elusive intermediates and greatly magnify the range of mechanistic questions that can be answered.     

X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature.

A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9 degrees C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-A resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.     

Key feature of the catalytic cycle of TNF-alpha converting enzyme involves communication between distal protein sites and the enzyme catalytic core

Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal-protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-alpha converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design.     

Binding of ligands to the active site of carboxypeptidase A.

We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH3OC6H4(CO)CH2CH(CO2(-))C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion of valine-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S1'. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S1', as a competitive inhibitor for esters (for which entry into S1' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S1' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S1', although not necessarily by the same chemical steps.     

Discovery of competing anaerobic and aerobic pathways in umpolung amide synthesis allows for site-selective amide 18O-labeling

The mechanism of umpolung amide synthesis was probed by interrogating potential sources for the oxygen of the product amide carbonyl that emanates from the α-bromo nitroalkane substrate. Using a series of (18)O-labeled substrates and reagents, evidence is gathered to advance two pathways from the putative tetrahedral intermediate. Under anaerobic conditions, a nitro-nitrite isomerization delivers the amide oxygen from nitro oxygen. The same homolytic nitro-carbon fragmentation can be diverted by capture of the carbon radical intermediate with oxygen gas (O(2)) to deliver the amide oxygen from O(2). This understanding was used to develop a straightforward protocol for the preparation of (18)O-labeled amides in peptides by simply performing the umpolung amide synthesis reaction under an atmosphere of 18O2.     

A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.

The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P'1 or P'2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.     

Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site

Aminopeptidase N from Escherichia coli is a major metalloprotease that participates in the controlled hydrolysis of peptides in the proteolytic pathway. Determination of the 870-aa structure reveals that it has four domains similar to the tricorn-interacting factor F3. The thermolysin-like active site is enclosed within a large cavity with a volume of 2,200 A(3), which is inaccessible to substrates except for a small opening of approximately 8-10 A. The substrate-based inhibitor bestatin binds to the protein with minimal changes, suggesting that this is the active form of the enzyme. The previously described structure of F3 had three distinct conformations that were described as "closed," "intermediate," and "open." The structure of aminopeptidase N from E. coli, however, is substantially more closed than any of these. Taken together, the results suggest that these proteases, which are involved in intracellular peptide degradation, prevent inadvertent hydrolysis of inappropriate substrates by enclosing the active site within a large cavity. There is also some evidence that the open form of the enzyme, which admits substrates, remains inactive until it adopts the closed form.     

Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1

A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.     

EFFECT OF D2O ON THE CARBOXYPEPTIDASE-CATALYZED HYDROLYSIS OF O-(trans-CINNAMOYL)-L-β-PHENYLLACTATE AND N-(N-BENZOYLGLYCYL)-L-PHENYLALANINE

Solvent isotope effects have been examined for the action of the zinc-containing metalloenzyme carboxypeptidase A on ester and peptide substrates. The kinetic parameters for the carboxypeptidase-catalyzed hydrolysis of an ester, O-(trans-cinnamoyl)-L-beta-phenyllactate, in 0.05 M Tris-DCl buffer containing 0.5 M NaCl at pD 8.07 and 25 degrees were compared with those obtained from measurements done in 0.05 M Tris-HCl buffer containing 0.5 M NaCl at pH 7.52 and 25 degrees . A (k(cat))(H2O)/(k(cat))(D2O) ratio of approximately 2 was obtained. The value of the Michaelis constant K(m) was unaffected by the change in solvent as was the inhibition constant, K(i), found for the product, L-beta-phenyllactate, which is a competitive inhibitor. These results indicate that a catalytic step involving general base catalysis is probably important in the carboxypeptidase-catalyzed hydrolysis of an ester. A similar set of experiments carried out on the peptide substrate, N-(N-benzoylglycyl)-L-phenylalanine gave ambiguous results. The role of the zinc ion in the catalytic action of carboxypeptidase A can be considered in the light of these findings.     

Amplified fluorescence sensing of protease activity with conjugated polyelectrolytes

Fluorescent conjugated polyelectrolytes with pendant ionic sulfonate and carboxylate groups are used to sense protease activity. Inclusion of the fluorescent conjugated polyelectrolyte into the assay scheme leads to amplification of the sensory response. The sensing mechanism relies on an electrostatic interaction between the conjugated polyelectrolyte and a peptide substrate that is labeled with a fluorescence quencher. Enzyme activity and hydrolysis kinetics are measured in real time by using fluorescence spectroscopy. Two approaches are presented. In the first approach, a fluorescence turn-on sensor was developed that is based on the use of p-nitroanilide-labeled peptide substrates. In this system enzyme-catalyzed peptide hydrolysis is signaled by an increase in the fluorescence from the conjugated polyelectrolyte. The turn-on system was used to sense peptidase and thrombin activity when the concentrations of the enzyme and substrate are in the nanomolar regime. Kinetic parameters were recovered from real-time assays. In the second approach, a fluorescence turn-off sensor was developed that relies on a peptide-derivatized rhodamine substrate. In the turn-off system enzyme-catalyzed peptide hydrolysis is signaled by a decrease in the fluorescence intensity of the conjugated polyelectrolyte.     

Substrate specificity of tonin from rat submaxillary gland.

The substrate specificity of tonin from rat submaxillary gland was examined with a series of synthetic peptides encompassing the C-terminus of the decapeptide substrate angiotensin I. In contrast to angiotensin I-converting enzyme from plasma or lung, only angiotensin I, (des-Asp1)-angiotensin I, and (des-Asp1, des-Arg2)-angiotensin I are substrates of tonin with Km values of 34.5 muM, 39.3 muM, and 54.4 muM, respectively, while the shorter C-terminal peptides are not hydrolyzed. Thus, the N-terminal sequence extending from position 1 to 3 is the enzymatic binding site for tonin. Turnover numbers of 33.4 sec-1, 42.8 sec-1, and 6.5 sec-1 are observed for the hydrolysis of angiotensin I, (des-Asp1)-angiotensin I, and (des-Asp1, des-Arg2)-angiotensin I, respectively. The relative percentage rates of hydrolysis (proportional to V/Km) at low substrate concentrations ([S] less than less than Km) are almost identical for (des-Asp1)-angiotensin I, angiotensin I, and the tetradecapeptide substrate, indicating that these three peptides are equally good substrates at low physiological concentrations. The observed high specificity of the enzyme lends support to the possible important role of tonin for local conversion in tissue. The conversion of (des-Asp1)-angiotensin I to (des-Asp1)-angiotensin II (angiotensin III) is of particular interest in relation to the recently suggested, potential role of the latter peptide in aldosterone release.     

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Two 29-residue peptides were prepared, one of which (ChPepz) was designed by surface-simulation synthesis to mimic the active site of alpha-chymotrypsin, and the other (TrPepz), which contained four substitutions relative to ChPepz, was fashioned after the active site of trypsin. Each peptide was cyclized by a disulfide bond. The ChPepz monomer effected hydrolysis of the ester group in N-benzoyl-L-tyrosine ethyl ester, an alpha-chymotrypsin substrate, with Km and kcat values that were comparable to those of alpha-chymotrypsin. ChPepz was completely inactivated by diisopropyl fluorophosphate (DIFP), L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK), or reduction of the disulfide bond. It had no catalytic activity on N-tosyl-L-arginine methyl ester, a trypsin substrate. On the other hand, TrPepz, which had no effect on N-benzoyl-L-tyrosine ethyl ester, hydrolyzed N-tosyl-L-arginine methyl ester with a Km value that was essentially identical to that of trypsin, but its kcat value was almost half that of trypsin. TrPepz was fully inactivated by reduction of the disulfide bond, by DIFP, or by phenylmethylsulfonyl fluoride but not by TPCK. It was also completely inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and human alpha 1-antitrypsin. ChPepz and TrPepz hydrolyzed proteins (myoglobin and casein) to give panels of peptides that were similar to those of the same protein obtained with the respective enzyme. However, TrPepz was more efficient than trypsin at hydrolyzing the C bonds of two or more consecutive lysine and/or arginine residues. Like its esterase activity, the proteolytic activity of ChPepz was inhibited by either DIFP or TPCK whereas that of TrPepz was inhibited by either DIFP or phenylmethylsulfonyl fluoride but not by TPCK. Finally, ChPepz and TrPepz were each more active at low temperature than the respective enzyme. This ability to construct fully functional peptide enzymes (pepzymes) of chosen specificities should find many practical applications.     

Site-directed mutagenesis and the role of the oxyanion hole in subtilisin

Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtilisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.     

Utilization of a depsipeptide substrate for trapping acyl—enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases

The penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus catalyzed the hydrolysis of the D-lactic acid residue from the depsipeptide diacetyl-L-lysyl-D-alanyl-D-lactic acid. The ester substrate was hydrolyzed faster than the peptide analogue, diacetyl-L-lysyl-D-alanyl-D-alanine, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, presumably because acylation (k(2)), which is the rate-limiting step of the peptidase reaction, occurred more rapidly during cleavage of the ester bond than during cleavage of the amide bond. No rate acceleration was observed with the S. aureus carboxypeptidase for which deacylation (k(3)) is already the rate-determining step with the peptide substrate. The efficiency of utilization of the depsipeptide (V(max)/K(m)) was greatly enhanced (19- to 147-fold) for all three enzymes. After incubation of the B. subtilis carboxypeptidase and [(14)C]diacetyl-L-lysyl-D-alanyl-D-lactic acid at pH 5.0 and lowering of the pH to 3.0, a radioactive acyl-enzyme intermediate containing 0.43 mol of substrate per mol of enzyme was isolated by Sephadex G-50 chromatography. After acetone precipitation, the acyl group of the denatured acyl-enzyme complex appeared to be bound to the protein by an ester bond. Acyl enzymes were also detected for the S. aureus and E. coli carboxypeptidases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography of enzyme incubated with [(14)C]depsipeptide and precipitated with acetone.     

Exploring the mechanism of protein synthesis with modified substrates and novel intermediate mimics

Translation, the synthesis of proteins from individual amino acids based on genetic information, is a cornerstone biological process. During ribosomal protein synthesis, new peptide bonds form through aminolysis of the peptidyl-tRNA ester bond by the alpha-amino group of the A-site amino acid. The rate of this reaction is accelerated at least 10(7)-fold in the ribosome, but the catalytic mechanism has remained controversial. We have used a combination of synthetic chemistry, biochemical, and structural biology approaches to characterize the mechanism of the peptidyl transfer reaction and the configuration of the reaction's tetrahedral intermediate. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. This indicates that the 2'-OH is essential to the reaction through participation in substrate assisted catalysis. A series of novel mimics of the tetrahedral intermediate were examined to distinguish between possible regio- and stereoisomeric forms of the intermediate. The determination of these parameters has important implications for the configuration of the substrates and intermediate within the ribosomal active site, and thus which functional groups are properly positioned to play various roles in promoting the reaction. Our results contribute to an emerging model of the peptidyl transfer reaction in which the ribosomal active site positions the substrates in an orientation specifically designed to promote the reaction, wherein the A76 2'-OH serves as a proton shuttle to enable critical proton transfers in the formation of the final peptide product.     

THE MECHANISM OF ACTION OF RIBONUCLEASE

The possible mechanisms of action of bovine pancreatic ribonuclease are discussed in the light of the detailed knowledge of the geometry of the active site that has been derived from studies of inhibitor binding by X-ray diffraction and nuclear magnetic resonance. When combined with a knowledge of the mechanism of phosphate ester hydrolysis, this information imposes severe geometric constraints on possible mechanisms of action of the enzyme. Two types of mechanism can be distinguished, the linear and the pseudorotation. The linear mechanism includes a catalytic role for both histidine residues at the active site and does not involve pseudorotation of the intermediate. In contrast, in the pseudorotation mechanism one histidine residue performs all the catalytic functions, while the other serves only to bind the phosphate anion; this necessarily involves pseudorotation of the intermediate and specific protonation of the leaving group by the enzyme.The mode of binding of the product of the reaction, cytidine-3'-monophosphate, has been elucidated by X-ray diffraction and nuclear magnetic resonance. If the substrate binds in an analogous way, only the linear mechanism is possible. This mechanism is described in detail.     

A novel assay for factor XIII based on cross-linking of synthetic peptides: analysis of different substrates.

A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.