Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M−1s−1 and 5.8 M−1s−1, respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme–substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme–substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzyme-catalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the free-flavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4–5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.

cAMP-response element modulator tau is a positive regulator of testis angiotensin converting enzyme transcription.

Testis angiotensin-converting enzyme (ACE) is a unique form of ACE, only produced by male germ cells, and results from a testis-specific promoter found within the ACE gene. We have investigated the role of cAMP-response element modulator (CREM)tau in testis ACE transcription. In gel shift experiments, testes nuclear proteins retard an oligonucleotide containing the cAMP-response element (CRE) found at position -55 in the testis ACE promoter. Anti-CREM antibody supershifts this complex. Competitive gel shift shows that recombinant CREM tau protein and testis nuclear proteins have a similar specificity of binding to the tests ACE CRE. Functional analysis using in vitro transcription and transfection studies also demonstrate that CREM tau protein is a transcriptional activator of the testis ACE promoter. Western blot analysis identifies CREM tau protein in the protein-DNA complex formed between nuclear proteins and the testis ACE CRE motif. This analysis also identified other CREM isoforms in the gel-shifted complex, which are thought to be CREM tau 1/2, CREM alpha/beta, and S-CREM. These data indicate that CREM tau isoforms play an important role as a positive regulator in the tissue-specific expression of testis ACE.

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: a possible coordinate role in ribosome biogenesis.

RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribonuclease originally described in the nucleus that processes RNA transcripts to generate their mature 5' termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2'-O-methyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized that RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.

A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

A plastid enzyme arrested in the step of precursor translocation in vivo.

The key enzyme of chlorophyll biosynthesis in higher plants, NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR, EC 1.3.1.33), accumulates in its precursor form (pPORA) in barley. pPORA is bound to the chloroplasts and is able to interact with the enzyme's substrate, Pchlide, at both the cytosolic as well as the stromal side of the plastid envelope. The interaction with intraplastidic Pchlide, formed in ATP-containing chloroplasts upon feeding with -aminolevulinic acid, drives vectorial translocation of pPORA across the plastid envelope membranes. In contrast, exogenously applied Pchlide causes the release of the envelope-bound precursor protein to the cytosol. Both processes compete with each other if intra- and extraplastidic Pchlide are applied simultaneously. A cytosolic heat shock cognate protein of Mr 70,000 present in wheat germ and barley leaf protein extracts appears to prevent the release of the pPORA to the cytosol in vivo, however.

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.

Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.

A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence. The cDNA encodes a 318-amino acid protein of Mr 35,960. The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase. In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence. There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme. Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates. With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product. The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate. Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity. Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate. These properties are consistent with the enzymes derived from human and rat sources.

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.