Elliott Shaw

D-PHE-PRO-ARGCH2Cl-A selective affinity label for thrombin

We had observed earlier that a D amino acid residue in the P/sub 3/ position, although not found in natural substrates, increased the effectiveness of an affinity label for thrombin. Thus, D-A1a-A1a-LysCH/sub 2/Cl was found to inactivate thrombin about 18 times more effectively than the L-isomer (5). The observation of Bajusz et al (6) that D-Phe-Pro-Arg-h, a tripeptide aldehyde analog, was a potent, reversible inhibitor of thrombin (K/sub i/ = 7.5 x 10/sup -8/M) suggested that phenylalanine in the P/sub 3/ position was particularly effective. We therefore extended our work to the synthesis of D-Phe-Pro-ArgCH/sub 2/C1 and report some preliminary observations of its unusual effectiveness and selectivity in the inactivation of thrombin. D-Phe-Pro-ArgCH/sub 2/C1 was prepared by the general procedure for the preparation of peptides of orginine chloromethyl ketone which we have described previously (7). The mixed anhydride of Z-D-Phe-Pro-OH was coupled to H-Arg(NO/sub 2/)CH/sub 2/Cl and the product was purified by chromatography on a silica gel column using chloroform: methanol (95:5) as a developing solvent to yield Z-D-Phe-Pro-Arg(NO/sub 2/)CH/sub 2/Cl.

Thiobenzyl benzyloxycarbonyl-l-lysinate, substrate for a sensitive colorimetric assay for trypsin-like enzymes

Abstract Thiobenzyl benzyloxycarbonyl- l -lysinate (Z-Lys-SBzl), a substrate for trypsin-likeproteases, was synthesized. In the presence of 5,5′-dithiobis(2-nitrobenzoic acid) the hydrolysis of the thiol ester by trypsin-like enzymes provides a continuous colorimetric assay with a sensitivity comparable to the best fluorometric substrates. Z-Lys-SBzl is readily synthesized in good yield, is water soluble, and has a low rate of spontaneous hydrolysis even at pH 8.0. This assay procedure has been routinely used with urokinase, human urinary and human plasma kallikrein, thrombin, plasmin, β -trypsin, factor Xa, and crotalase. Levels of detection of these enzymes are in the range 10 −14 to 10 −13 mol.

Human plasma kallikrein: Purification and preliminary characterization☆

Abstract A method is described for the convenient purification of the protease plasma kallikrein from human Cohn fraction IV-1. The enzyme was produced by endogenous activation after acid treatment to remove an inhibitor and was concentrated by the successive use of affinity adsorbents prepared by the immobilization of soybean trypsin inhibitor and aminobenzamidine. The esterase- and kinin-producing activities were enriched about 1100-fold from fraction IV-1. Several properties of plasma kallikrein strengthen the impression that it is related to trypsin, namely, competitive inhibition by benzamidine and the formation of a stable p-guanidinobenzoyl acyl enzyme intermediate. Inactivation by affinity labeling with Z-LysCH2Cl was successful in contrast to the inertness of Tos-LysCH2Cl.

Rapid inactivation of cathepsin L by Z-Phe-PheCHN2 and Z-Phe-AlaCHN2

Z-Phe-PheCHN/sub 2/ and Z-Phe-AlaCHN/sub 2/ were found to react extremely rapidly with cathepsin L from rat liver lysosomes. For measuring the inactivation time it was necessary to work with dilute enzyme and inhibitor solutions, that is in the range of 10/sup -9/ M. These conditions were made possible through the use of Z-Phe-Arg-4-methyl-7-coumarylamide, a very sensitive substrate suitable for assays of cathepsin L in about 10/sup -12/ M solutions. Z-Phe-AlaCHN/sub 2/ has an affinity for cathepsin L which is about 2000-fold higher than for cathepsin B from rat and human. Z-Phe-PheCHN/sub 2/ proved to be a selective inactivator of cathepsin L in a certain concentration range. Z-Phe-PheCHN/sub 2/ reacts reversibly with cathepsin B from several species.

Further observations on substrate-derived chloromethyl ketones that inactivate trypsin

Abstract The inactivation of trypsin by TLCK was examined with respect to dependence on the concentration of the inhibitor. Saturation kinetics were observed as expected of an active-site directed reagent. The intermediate complex has a dissociation constant of 2.1 × 10 −4 m and forms inactive enzyme with K 2 = 0.16 min −1 at pH 7.0, 25 °. An improved synthesis of TLCK is described which also facilitated the preparation of D-TLCK and of LCK (the chloromethyl ketone derived from lysine). The former is essentially inactive. LCK alkylates trypsin at N-3 of a histidine residue. Although it has less affinity for trypsin than TLCK, LCK forms a complex which proceeds to alkylated enzyme more rapidly, i.e., k 2 = 0.50 min −1 . The inactivation of trypsin by TLCK was examined in the region of pH 4.0–4.4 at 37 °. Under these conditions considerable denaturation was occurring. However, alkylation took place only with active enzyme, exclusively at N-3 of a histidine residue. Attempts made to synthesize chloromethyl ketones derived from N α -substituted ornithine and arginine encountered difficulty chiefly due to cyclization. However, preparations containing a low content of the chloromethyl ketone from N α - p -nitrobenzyloxycarbonylarginine ( p -NO 2 -ZACK) were obtained; this agent is an extremely rapid inactivator of trypsin.

Active center differences between cathepsins L and B: The S1 binding region

The substrate peptide bond cleaved by cathepsins B and L is determined not by the amino acid contributing the carboxyl group to this bond as in the case of serine proteases but rather by the presence of a neighboring amino acid with a large hydrophobic side chain. From a study of the inhibitory potency in a series, Cbz‐Phe‐X‐CHN2, in which Phe promotes binding at S2 (terminology of [(1968) Biochem. Biophys. Res. Commun. 32, 898–902]) while the amino acid X probes S1, it is shown that this region of cathepsin L also has the ability to accommodate large hydrophobic side chains. In this respect cathepsin L differs from cathepsin B. Thus Cbz‐Phe‐Tyr(O‐t‐Bu)CHN2 inactivates cathepsin L with a rate 2.5 × 104 greater than that for cathepsin B.

The reactivity of His-57 in chymotrypsin to alkylation.

Variations in the structure of TPCK, the chloromethylketone derived from tosylphenylalanine have been examined as inactivators of chymotrypsin. An N α -acyl group is not essential for the exclusive alkylation of His-57 in chymotrypsin, but elimination of the α-amino group has been shown to favor methionine modification. The nature of the N α -acyl substituent has a considerable effect on the rate of inactivation which, in the case of the benzyloxycarbonyl derivatives ZPCK and ZPBK, is about 10 6 -fold greater with His-57 in chymotrypsin than with acetyl histidine as a model.

The affinity-labelling of cathepsin s with peptidyl diazomethyl ketones: Comparison with the inhibition of cathepsin L and calpain

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z‐Leu‐Leu‐Nle‐CHN2 inactivates cathepsin S with k 2nd = 4.6 × 106 M−1.s−1 at pH 6.5,25°C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z‐Val‐Val‐NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z‐Phe‐Tyr(t‐Bu)CHN2 is about 104 more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.

A comparison of the behavior of chymotrypsin and cathepsin B towards peptidyl diazomethyl ketones

Chymotrypsin is not inactivated by benzyloxycarbonyl-phenylalanyl diazomethyl ketone although disappearance of the diazo group can be followed spectroscopically. It is also inert to various dipeptide derivatives. Cathepsin B on the other hand is inactivated by this reagent, as described earlier as well as by other peptidyl diazomethyl ketones. It appears from initial studies that a phenylalanyl residue in the penultimate position of the inhibitor is favorable for effectiveness. Benzyloxycarbonyl-Phe-AlaCHN/sub 2/ emerges from this work as a powerful, relatively soluble inactivator of bovine spleen cathepsin B with K/sub i/ = 1.7 x 10/sup -6/M.

An exploration of the primary specificity site of cathepsin b

Peptidyl diazomethyl ketones inactivate cathepsin B apparently by alkylation of the active center thiol following complex formation as in the case of benzyloxycarbonyl (Cbz)-Phe-AlaCHN2. The phenylalanine contributes considerably to binding in the secondary specificity site. In order to define the topography of the active center region comprising the primary specificity site of beef spleen cathepsin B, a series of peptidyl diazomethyl ketones having the general structure Cbz-Phe-X-CHN2 has now been synthesized. The amino acid, X, has been varied in size to include rather large side chains which might reveal available binding potential or limitations. Some of the reagents, in fact, were not inhibitory even at 10(-4) M. Others, however, that did measurably inactivate cathepsin B provided a range of reactivities that extended over 5 orders of magnitude and correlated with affinity in the reversible phase of inactivation. Some large side chains, for example, that of tryptophan, were very poorly tolerated in this region of the active center, whereas others, such as O-benzyl threonine, provided remarkably active inhibitors. A topographical rationalization of the results is offered.