The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin.

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., Ki) and the overall inhibition constants (i.e., Ki*) were calculated to be 11.5 microM and 0.4+/-0.14 nM, respectively. The second-order rate constant for the reaction was 4.5+/-0.19x10(5) M(-1)s(-1), and the product released following the acylation step, 6-amidino2-naphthol, showed mixed-type inhibition. The competitive (Kic) and uncompetitive (Kiu) inhibition constants were 14.7 microM and 19.5 microM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s(-1) and 195 s(-1). The deacylation step was relatively much slower (3.2+/-0.19x10(-5) s(-1), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 microM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.

The use of fluorogenic substrates to monitor thrombin generation for the analysis of plasma and whole blood coagulation.

Thrombin is central to the process of coagulation and monitoring its activity is a reliable indicator of the rate and extent of coagulation. I have employed a range of fluorogenic peptide substrates as indicators of coagulation via the formation of active thrombin. This system enabled coagulation to be monitored in a kinetic fashion, and the use of fluorescence enabled a wide range of samples to be analyzed including lyophilized plasma containing fibrin, fresh platelet-poor plasma, platelet-rich plasma, and even whole blood. Coagulation could be monitored following triggering by tissue factor, ellagic acid, or each of the proteases preceding thrombin in the coagulation network. Using this assay procedure I have investigated the anticoagulant activities of a number of compounds and the results indicate that this assay would be useful for the kinetic analysis of coagulation in various plasma preparations, or even whole blood.

Crystal structure of aspartate decarboxylase at 2.2 A resolution provides evidence for an ester in protein self-processing.

The structure of L-aspartate-alpha-decarboxylase from E. coli has been determined at 2.2 A resolution. The enzyme is a tetramer with pseudofour-fold rotational symmetry. The subunits are six-stranded beta-barrels capped by small alpha-helices at each end. The active sites are located between adjacent subunits. The electron density provides evidence for catalytic pyruvoyl groups at three active sites and an ester at the fourth. The ester is an intermediate in the autocatalytic self-processing leading to formation of the pyruvoyl group. This unprecedented structure provides novel insights into the general phenomenon of protein processing.

Escherichia coli L-aspartate-alpha-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry.

The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was cloned by PCR, and shown to complement a panD mutant defective in beta-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the pi-protein), which is proteolytically cleaved at a specific X-Ser bond to produce a beta-subunit with XOH at its C-terminus and an alpha-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed pi-subunit (of 13.8 kDa), with a small proportion of the alpha- and beta-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the alpha-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151+/-16 microM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain.

A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain. The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae. The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l. The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences. Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant. Two different types of affinity chromatography were used to purify either propapain or papain. The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously. The kinetic parameters obtained agree well with the literature values. The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems. This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.

Escherichia coli chorismate synthase catalyzes the conversion of (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate to 6-fluorochorismate. Implications for the enzyme mechanism and the antimicrobial action of (6S)-6-fluoroshikimate.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh enzyme of the shikimate pathway, which is responsible for the biosynthesis of aromatic metabolites from glucose. The chorismate synthase reaction involves a 1,4-elimination with unusual anti-stereochemistry and requires a reduced flavin cofactor. The substrate analogue (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate is a competitive inhibitor of Neurospora crassa chorismate synthase (Balasubramanian, S., Davies, G. M., Coggins, J. R., and Abell, C. (1991) J. Am. Chem. Soc. 113, 8945-8946). We have shown that this analogue is converted to 6-fluorochorismate by Escherichia coli chorismate synthase at a rate 2 orders of magnitude slower than the normal substrate. The decreased rate of reaction is consistent with the destabilization of an allylic cationic intermediate. The formation of chorismate and 6-fluorochorismate involves a common protein-bound flavin intermediate although the fluoro substituent does influence the spectral characteristics of this intermediate. The fluoro substituent also decreased the rate of decay of the flavin intermediate by 280 times. These results are consistent with the antimicrobial activity of (6S)-6-fluoroshikimate not being mediated by the inhibition of chorismate synthase but by the inhibition of 4-aminobenzoic acid synthesis as previously proposed (Davies, G. M., Barrett-Bee, K. J., Jude, D. A., Lehan, M., Nichols, W. W., Pinder, P. E., Thain, J. L., Watkins, W. J., and Wilson, R. G. (1994) Antimicrobial Agents and Chemotherapy 38, 403-406).

A continuous, anaerobic spectrophotometric assay for chorismate synthase activity that utilizes photoreduced flavin mononucleotide.

A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of +/- 2 microM product. The assay conditions have been optimized with respect to FMNH2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.