Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.

Hepatitis C virus NS3/4A protease.

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.

Hepatitis C virus NS3/4A protease.

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.

Metabolism of amprenavir in liver microsomes: role of CYP3A4 inhibition for drug interactions.

Amprenavir (141W94, VX-478, KVX-478) is metabolized primarily by CYP3A4 (cytochrome P450 3A4) in recombinant systems and human liver microsomes (HLM). The effects of ketoconazole, terfenadine, astemizole, rifampicin, methadone, and rifabutin upon amprenavir metabolism were examined in vitro using HLM. Ketoconazole, terfenadine, and astemizole were observed to inhibit amprenavir depletion, consistent with their known specificity for CYP3A4. The HIV protease inhibitors, indinavir, saquinavir, ritonavir, and nelfinavir, were included in incubations containing amprenavir to examine the interactions of HIV protease inhibitors in vitro. The order of amprenavir metabolism inhibition in human liver microsomes was observed to be: ritonavir > indinavir > nelfinavir > saquinavir. The Ki value for amprenavir-mediated inhibition of testosterone hydroxylation in human liver microsomes was found to be approximately 0.5 microM. Studies suggest that amprenavir inhibits CYP3A4 to a greater extent than saquinavir, and to a much lesser extent than ritonavir. Amprenavir, nelfinavir, and indinavir appear to inhibit CYP3A4 to a moderate extent, suggesting a selected number of coadministration restrictions.

Mechanistic role of an NS4A peptide cofactor with the truncated NS3 protease of hepatitis C virus: elucidation of the NS4A stimulatory effect via kinetic analysis and inhibitor mapping.

Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343-355] delineates a small hydrophobic region within the 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addresses the mechanism of NS3 protease catalytic activation by NS4A utilizing a small synthetic NS4A peptide (residues 1678-1691 of the virus polyprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 kcat and kcat/Km catalytic parameters when measured against small peptide substrates representing the different site-specific junctions of the polyprotein. The catalytic effect of natural and non-natural amino acid substitutions at the P1 position in a 5A/5B peptide substrate was investigated. NS3-NS4A demonstrated a marked catalytic preference for the cysteine residue commonly found in authentic substrates. The pH dependence of the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pKa values of the active site residues of NS3 protease. A steady state kinetic analysis was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, with NS4A binding first. Two distinct kinetic classes of peptidyl inhibitors based upon the 5A/5B cleavage site were identified. An NS4A-independent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P1' residue serine. These inhibitors display an up to 80-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residues from this inhibitor class demonstrated the fact that the P4' and P1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide substrate bound to the active site of the NS3-NS4A structure.

Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases.

Interleukin-1beta-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1beta (pre-IL-1beta) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M-1 s-1 for CPP32 to 1.0 x 10(3) M-1 s-1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.

Expression and purification of human interleukin-1beta converting enzyme from Trichoplusia ni insect cells using a baculovirus expression system.

Employing a baculovirus expression system, human interleukin-1beta converting enzyme (ICE) has been expressed in Trichoplusia ni High-Five insect cells and purified. ICE was expressed with an N-terminal T7 epitope, thus allowing its purification using immobilized anti-T7 antibodies. The recombinant ICE was purified to >95% homogeneity, the one minor contaminant being the baculovirus anti-apoptotic protein (P35) which was copurified. The purified recombinant ICE was biologically active, cleaving both pIL-1beta and poly(ADP-ribose) polymerase substrates. The kinetic properties of the purified recombinant ICE compare favorably with native ICE purified from a THP-1 monocytic line.

Structure and mechanism of inosine monophosphate dehydrogenase in complex with the immunosuppressant mycophenolic acid.

The structure of inosine-5'-monophosphate dehydrogenase (IMPDH) in complex with IMP and mycophenolic acid (MPA) has been determined by X-ray diffraction. IMPDH plays a central role in B and T lymphocyte replication. MPA is a potent IMPDH inhibitor and the active metabolite of an immunosuppressive drug recently approved for the treatment of allograft rejection. IMPDH comprises two domains: a core domain, which is an alpha/beta barrel and contains the active site, and a flanking domain. The complex, in combination with mutagenesis and kinetic data, provides a structural basis for understanding the mechanism of IMPDH activity and indicates that MPA inhibits IMPDH by acting as a replacement for the nicotinamide portion of the nicotinamide adenine dinucleotide cofactor and a catalytic water molecule.

Structure and mechanism of interleukin-1 beta converting enzyme.

Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

Microbes and microbial enzymes for cyanide degradation.

Cyanide is an important industrial chemical produced on a grand scale each year. Although extremely toxic to mammalian life, cyanide is a natural product generated by fungi and bacteria, and as a result microbial systems have evolved for the degradation of cyanide to less toxic compounds. The enzymes which utilize cyanide as a substrate can be categorized into the following reaction types: substitution/addition, hydrolysis, oxidation, and reduction. Each of these categories is reviewed with respect to the known biochemistry and feasibility for use in treatment of cyanide containing wastes.

Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.

Kinetic characterization of the [3'-32P]coenzyme A/acetyl coenzyme A exchange catalyzed by a three-subunit form of the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum.

The ability of acetyl coenzyme A synthesizing carbon monoxide dehydrogenase isolated from Clostridium thermoaceticum to catalyze the exchange of [3'-32P]coenzyme A with acetyl coenzyme A is studied. This exchange is found to have a rate exceeding that of the acetyl coenzyme A carbonyl exchange also catalyzed by CO dehydrogenase ([1-14C]acetyl coenzyme A + CO in equilibrium acetyl coenzyme A + 14CO). These two exchanges are diagnostic of the ability of CO dehydrogenase to synthesize acetyl coenzyme A from a methyl group, coenzyme A, and carbon monoxide. The kinetic parameters for the coenzyme A exchange have been determined: Km(acetyl coenzyme A) = 1500 microM, Km(coenzyme A) = 50 microM, and Vmax = 2.5 mumol min-1 mg-1. Propionyl coenzyme A is shown to be a substrate (Km approximately 5 mM) for the coenzyme A exchange, with a rate 1/15 that of acetyl coenzyme A, but is not a substrate for the carbonyl exchange. CO dehydrogenase capable of catalyzing both these two exchanges, and the oxidation of CO to CO2, is isolated as a complex of molecular weight 410,000 consisting of three proteins in an alpha 2 beta 2 gamma 2 stoichiometry. The proposed gamma subunit, not previously reported as part of CO dehydrogenase, copurifies with the enzyme and has the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the disulfide reductase previously separated from CO dehydrogenase in a final chromatographic step.

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum.

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.

Epoxidation of olefins by cytochrome P-450 model compounds: mechanism of oxygen atom transfer.

The mechanism of the Mn(III) porphyrin-catalyzed epoxidation of olefins by lithium hypochlorite is examined. The active oxidant is thought to be a high-valent manganese-oxo complex. It is shown that a relatively stable intermediate is reversibly formed upon interaction of the olefin and the oxo complex. The decomposition of this intermediate to Mn(III) porphyrin and epoxide is the rate-determining step of the catalytic cycle. Some analogies to the biochemical epoxidation of olefins catalyzed by cytochrome P-450 are discussed.

Oxygenation of hydrocarbons by cytochrome P-450 model compounds: modification of reactivity by axial ligands.

The rate of olefin oxygenation catalyzed by synthetic metalloporphyrins is examined, employing sodium hypochlorite as the oxygen atom source. The rate of epoxidation and the stability of the catalyst are shown to be dependent on the nature of the axial ligand employed. A rationale for this effect is presented and analogy is made to the role of the thiolate ligand in cytochrome P-450.