Converting trypsin to chymotrypsin: the role of surface loops.

Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate, but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and substrate discrimination occurs during the acylation step rather than in substrate binding.

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure.

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.

Guanidine derivatives restore activity to carboxypeptidase lacking arginine-127.

Arg-127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn2+ carboxypeptidase A-catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg-127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in kcat (kcat/Km is decreased by 10(4) for R127A). Therefore, Arg-127 stabilizes the enzyme-transition state complex but not the ground state enzyme-substrate complex (Phillips, M.A., Fletterick, R., & Rutter, W.J., 1990, J. Biol. Chem. 265, 20692-20698). The addition of guandine, methylguanidine, or ethylguanidine to R127A increases the kcat for hydrolysis of Bz-gly(o)phe by 10(2) without changing the Km. Dissociation constants (Kd) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz-phe-alaP(o)ala is increased similarly by 10(2); in contrast, the binding affinity of the ground state inhibitor benzylsuccinic acid is not altered. Thus, guanidine derivatives mimic Arg-127 in stabilizing the rate-limiting transition state. Hydrolysis of Bz-gly-(o)phe by wild-type carboxypeptidase, R127K, or R127M is not substantially affected by guanidine derivatives. Additionally, primary amines do not change the activity of R127A. These observations imply that guanidine binds in the cavity vacated by Arg-127 specifically and in a productive conformation for catalysis.

The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity.

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.

IMP dehydrogenase: mechanism of action and inhibition.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the concomitant reduction of NAD to NADH. This reaction is the rate-limiting step in guanine nucleotide biosynthesis. IMPDH is a proven target for immunosuppressive, anticancer and antiviral chemotherapy, and may also be a target for antimicrobial agents. IMPDH is activated by monovalent cations, and one monovalent cation binding site appears to have been identified. The mechanism of IMPDH involves formation and hydrolysis of a covalent enzyme intermediate (E-XMP*) in a reaction reminiscent of glyceraldehyde-3-phosphate dehydrogenase. Substrates bind to IMPDH in a random order, hydride transfer is fast and NADH release precedes hydrolysis of E-XMP*. The hydrolysis of E-XMP* is at least partially rate-limiting. Two inhibitors, mizoribine-monophosphate and a fat base nucleotide appear to act as transition state analogs. In contrast, MPA inhibits by sequestering E-XMP.

Purine base transport in wild-type and mycophenolic acid-resistant Tritrichomonas foetus.

The purine base transport systems of wild-type and mycophenolic acid-resistant (MPAR) Tritrichomonas foetus have been characterized. Wild-type T. foetus has two carriers, one for hypoxanthine (Km = 0.7 +/- 0.3 mM, Vm = 80 +/- 20 pmol microliters-1min-1) and guanine (Km = 0.09 +/- 0.02 mM, Vm = 17 +/- 3 pmol microliters-1min-1), and a second for xanthine (Km = 0.6 +/- 0.2 mM, Vm = 25 +/- 5 pmol microliters-1min-1). Adenine transport was not saturable (k = 0.16 +/- 0.01 min-1) and therefore appears to enter the parasite by passive diffusion through the membrane. T. foetus MPAR has lost the hypoxanthine/guanine transporter. Xanthine and adenine transport are similar in wild-type and MPAR T. foetus. No purine nucleoside transporter could be identified.

Purification, characterization, and kinetic analysis of inosine 5'-monophosphate dehydrogenase of Tritrichomonas foetus.

The IMP dehydrogenase of Tritrichomonas foetus, a parasitic protozoan incapable of de novo biosynthesis of purine nucleotides, has been purified about 1000-fold to apparent homogeneity. The purified enzyme demonstrated a 20-fold higher substrate turnover rate than the pure IMP dehydrogenase from sarcoma ascites tumor cells. It has a subunit molecular weight of 58,000, aggregates to a size of 380,000 at low ionic strength, and partly dissociates to a molecular weight of 270,000 in high salt concentrations. Unlike the IMP dehydrogenase of bacteria and mammals, the T. foetus enzyme does not require K+ for activity. The analysis of initial velocity and product inhibition data is consistent with a sequential, ordered bi bi kinetic mechanism for the parasite enzyme-catalyzed reaction, in which IMP binds before NAD+ and NADH is released before XMP. This is in contrast to the partially random mechanism of the bacterial enzyme which involves the formation of an enzyme-K+-(IMP) complex. Mycophenolic acid inhibits T. foetus IMP dehydrogenase uncompetitively versus both IMP and NAD+ with an apparent Ki of 9 microM. This value, which is several hundred-fold higher than that for mammalian IMP dehydrogenase, suggests significantly different binding properties of the mycophenolic acid site in T. foetus IMP dehydrogenase, which might be amenable to specific inhibitor design.

A novel mechanism of mycophenolic acid resistance in the protozoan parasite Tritrichomonas foetus.

Tritrichomonas foetus relies primarily on the salvage of hypoxanthine to supply purine nucleotides. Mycophenolic acid disrupts T. foetus growth by specifically inhibiting inosine-5'-monophosphate (IMP) dehydrogenase, thereby blocking the biosynthesis of guanine nucleotides from hypoxanthine. We have cloned a T. foetus strain (mpar) that was 50-fold more resistant to mycophenolic acid than wild type (IC50 = 1 mM for mpar vs 20 microM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpar was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpar strains. Mycophenolic acid (100 microM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T. foetus mpar, although no inhibition of T. foetus mpar growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpar so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T. foetus mpar incorporated xanthine more efficiently into the nucleotide pool relative to hypoxanthine and guanine than wild type. Xanthine incorporation via XMP provided an IMP dehydrogenase independent route to guanine nucleotides that would enable the parasite to become mycophenolic acid resistant. No difference could be detected between wild type and mpar hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpar: it was deficient in hypoxanthine transport and had diminished adenine deaminase activity. The apparent net result of these two changes was to lower the intracellular concentration of hypoxanthine in mpar. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpar. The mpar strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.

Mortality in two cohorts of welders exposed to high- and low-levels of hexavalent chromium.

Hexavalent chromium particles are generated in the welding of stainless steel. These particles have manifested a mutagenic action in bacterial test systems and produced chromosome aberrations in cultured Chinese hamster cells. A cohort consisting of 234 welders working on stainless steel and exposed to high levels of chromium was selected. According to an earlier survey the hexavalent chromium exposure of such welders was often above 20 micrograms/m3. Another cohort consisting of 208 railway track welders exposed to low levels of chromium was also selected. The participants of both cohorts had welded for at least five years some time between 1950 and 1965 and were followed for mortality until December 1984. Among the welders exposed to high chromium levels five deaths occurred due to pulmonary tumors. This number is significantly greater than the one death that occurred among the welders exposed to low levels of chromium, but not significantly greater than the corresponding mortality of the general population. Thus exposure to stainless steel welding fumes might be associated with an increased incidence of pulmonary tumors.

Exposure and urinary excretion of aluminum during welding.

The exposure and urinary excretion of aluminum was studied among three previously unexposed volunteers and six welders exposed to welding fumes containing aluminum. The aluminum concentrations in air and urine were determined. The urinary aluminum concentrations rose rapidly in volunteers exposed only for 1 d and returned to the preexposure levels with an estimated half-time of about 8 h. The welders were monitored for one workweek. During the subsequent weekend a decrease in the urinary concentrations occurred in the three welders exposed for two years or less, but such a decrease was not observed among welders exposed for more than 15 years. The urinary concentrations of aluminum were dependent partly on the level of current exposure and partly on the duration of exposure. The data suggest that welders exposed to welding fumes containing aluminum may retain some of the inhaled metal fume for extended periods of time.

Work with video display terminals among office employees. I. Subjective symptoms and discomfort.

Subjective symptoms and discomfort were evaluated by means of a questionnaire and compared between approximately 400 video display terminal (VDT) operators and 150 selected referents. Previous and current illnesses, educational status, and smoking and drinking habits were also studied. The results showed the VDT operators to have more eye discomfort and possibly also more musculoskeletal discomfort in the shoulders, neck, and back than the referents. The VDT group also reported more skin disorders. In addition, women reported consistently more disorders than men, regardless of whether or not they were employed in VDT work. Women in general displayed greater morbidity than men. Eye discomfort, musculoskeletal discomfort, headache, and skin disorders were found to be significantly correlated in the material. The results also indicated that total daily workhours and time spent looking at the VDT screen were related to the degree of discomfort. Even when the subjects were divided into subgroups with reference to the various enterprises, the types of work and the makes of VDT, the differences obtained in the degree of discomfort appeared to be due to variations in the length of workhours.

Metronidazole for the prevention of dry socket after removal of partially impacted mandibular third molar: a randomised controlled trial.

We randomised 119 patients who had been referred for removal of partially impacted mandibular third molars to be given either metronidazole 1600 mg or placebo as a single dose 45 min before operation. Ten of the fifty-nine patients who were given metronidazole and 13 of the 60 given placebo developed dry sockets. Two variables were significantly associated with the development of a dry socket: pericoronitis and oral contraceptives.

The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the first committed step of guanosine 5'-monophosphate (GMP) biosynthesis, and thus regulates the guanine nucleotide pool, which in turn governs proliferation. Human IMPDHs are validated targets for immunosuppressive, antiviral and anticancer drugs, but as yet microbial IMPDHs have not been exploited in antimicrobial chemotherapy. Selective inhibitors of IMPDH from Cryptosporidium parvum have recently been discovered that display anti-parasitic activity in cell culture models of infection. X-ray crystal structure and mutagenesis experiments identified the structural features that determine inhibitor susceptibility. These features are found in IMPDHs from a wide variety of pathogenic bacteria, including select agents and multiply drug resistant strains. A second generation inhibitor displays antibacterial activity against Helicobacter pylori, demonstrating the antibiotic potential of IMPDH inhibitors.

The relation between bone mineral density in the heel and pixel intensity in the mandibular jaw bone among elderly women.

OBJECTIVES: The purpose of this study was to investigate the correlation between pixel intensity (PI) in digital radiographs of the lower jaw and bone mineral density (BMD) in the heels of post-menopausal women (as measured with DXL, a combination of dual energy X-ray absorptiometry and lasers). METHODS: Two intraoral periapical digital radiographs were taken in the right and left mandible premolar region, and the digital images were analysed by a computer program (Dimaxis) regarding PI. As the radiographs were taken, the BMD of the patient's left heel was measured via a portable Calscan device. The patient answered a questionnaire concerning risk factors. The correlation between variables was analysed using statistical tests. RESULTS: A significant correlation was found between the PI in the left (P = 0.001) and right (P = 0.004) mandible and the BMD of the left heel for the whole group. A pronounced correlation was found to exist for women > 70 years old. Based on a cut-off value of the PI, to differentiate between healthy individuals and those who required further analysis for osteoporosis, the following values were obtained: sensitivity 0.74, specificity 0.50, positive predictive value 0.77 and negative predictive value 0.46. CONCLUSION: A positive correlation was found between PI in digital radiographs of the mandible and the BMD of the heel. The low predictive value does not allow any definite conclusions to be drawn from the present study. A reasonable recommendation could be for future studies to employ a larger study population to explore the effect on this value.

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

BACKGROUND: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. OBJECTIVE: We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. METHODS AND RESULTS: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. CONCLUSION: These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

Trypsin: a case study in the structural determinants of enzyme specificity.

Trypsin and chymotrypsin have similar tertiary structures, although very different substrate specificities. Trypsin hydrolyzes peptides at Lys/Arg residues while chymotrypsin recognizes large hydrophobic residues. Recent work has shown that trypsin is not converted into a protease with chymotrypsin-like activity when the S1 substrate binding site residues are replaced with their chymotrypsin counterparts. Chymotrypsin-like activity is reconstituted in the trypsin framework when two surface loops are substituted with the analogous loops of chymotrypsin in addition to the substitutions at the S1 site. Chymotrypsin-like activity is further improved when Tyr172, another residue located outside the S1 site, is replaced with Trp. These mutant enzymes catalyze the hydrolysis of enzyme-bound substrate at rates comparable to chymotrypsin, but are defective in substrate binding. X-ray crystal structures of the chymotrypsin-like mutants reveal that the loops are disordered and that the Tyr172 to Trp substitution stabilizes the loops. These observations demonstrate that substrate specificity is derived from a network of structural interactions which extends beyond the S1 site.

Kinetic mechanism of human inosine 5'-monophosphate dehydrogenase type II: random addition of substrates and ordered release of products.

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD to NADH. This reaction is the rate-limiting step of guanine nucleotide biosynthesis. IMPDH is a target of immunosuppressive, antiviral, anticancer, and antiparasitic chemotherapy. We have determined a minimal kinetic mechanism for human IMPDH type II using NAD analogs, isotope effects, hydride exchange, and presteady state kinetics. The values of kcat for the NAD analogs are similar despite a great variation in the structure and reactivity of the compounds. This observation suggests that a common step is rate-limiting, i.e., either hydrolysis of the E-XMP* intermediate or release of XMP. No Vm isotope effect is observed when 2-2H-IMP is the substrate, which indicates that hydride transfer is fast. This conclusion is confirmed by the observation of a burst of NADH production under presteady state conditions. These observations further suggest that either E-XMP* hydrolysis or XMP release is rate-limiting. V/Km deuterium isotope effects are observed for both substrates (1.9 for IMP and 2.5 for NAD), which indicates that substrate association is random. This result contradicts previous conclusions based on product inhibition studies. No NADH consumption is observed in the presence of XMP and IMPDH, which indicates that the overall reaction is irreversible. NADH consumption is observed in the presence of thio-NAD, IMP, and enzyme. These observations indicate that NADH traps the E-XMP* intermediate and demonstrates that hydride transfer is reversible. At infinite NADH, all of E-XMP* is trapped by NADH, as indicated by the equivalence of the rates of consumption of thio-NAD and NADH. Therefore the release of products is ordered, with NADH release preceding hydrolysis of E-XMP*.

A potent "fat base" nucleotide inhibitor of IMP dehydrogenase.

Inosine monophosphate dehydrogenase (IMPDH) is a target for anticancer, antiviral, immunosuppressive, and antimicrobial chemotherapy. Thus, IMPDH inhibitors have great potential as chemotherapeutic agents. Here we show that imidazo[4,5-e][1, 4]diazapine nucleotide (I) is a potent inhibitor of both human type II and Escherichia coli IMPDH. I is a slow-binding inhibitor. The values of Kd are 1.4 nM and 53 nM for human and E. coli IMPDH, respectively. Inhibition is reversible, as demonstrated by the recovery of activity upon denaturation and renaturation of the enzyme.I complex. I is not a substrate for IMPDH. I may form a covalent adduct with the active-site Cys of IMPDH. Such an adduct would serve as an analogue for an intermediate in the IMPDH reaction.

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate.

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.