Biomedical subjects

C E Catalano

Publications and source records attributed to C E Catalano.

15 recordsLinked to original sources

PubMed1997

Kinetic characterization of the strand separation ("helicase") activity of the DNA packaging enzyme from bacteriophage lambda.

Bacteriophage lambda is assembled from preformed viral capsids (proheads), tails, and genomes that are excised from a concatemeric DNA precursor. The enzyme responsible for insertion of the genome into the precapsid is known as terminase. This enzyme possesses site-specific endonuclease, ATPase, and DNA strand separation ("helicase") catalytic activities, which work in concert to excise and package a single viral genome during phage assembly. We have previously characterized the endonuclease [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065] and ATPase [Tomka, M. A., & Catalano, C. E. (1993) Biochemistry 32, 11992-11997] catalytic activities of lambda terminase and present here similar studies on the strand separation activity of the enzyme. Strand separation requires terminase, divalent metal, and adenosine nucleotides with a hydrolyzable beta,gamma-phosphate bond. Two apparent binding sites for ATP-mediated strand separation were identified, one of which appears to be distinct from the high- and low-affinity sites previously observed for ATP hydrolysis [Hwang, Y., Catalano, C. E., & Feiss, M. (1995) Biochemistry 35, 2796-2803]. Salt stimulates the reaction at low concentrations but is strongly inhibitory at elevated concentrations, presumably due to impaired DNA binding. The above results are identical with either a complex DNA mixture (a nicked, annealed DNA duplex in the presence of excess nonspecific DNA) or a purified DNA substrate; however, a kinetic analysis of the reaction revealed that the observed rate was approximately 5-fold greater with the purified DNA substrate. Moreover, while Escherichia coli integration host factor (IHF) stimulates terminase-mediated strand separation with both substrates, the observed stimulation is more pronounced with the complex DNA mixture (10-fold rate increase) than the purified DNA substrate (5-fold rate increase). Our data are consistent with a model where IHF binding to the terminase assembly site forms a binary protein.DNA complex readily distinguishable from bulk DNA. The implications of these results to the process of DNA packaging in bacteriophage lambda are discussed.

Adenosine Triphosphatases↗

PubMed1997

Kinetic analysis of the endonuclease activity of phage lambda terminase: assembly of a catalytically competent nicking complex is rate-limiting.

The terminase enzyme from bacteriophage lambda is responsible for excision of a single genome from a concatameric DNA precursor and its insertion into an empty viral procapsid. The enzyme possesses a site-specific endonuclease activity which is responsible for excision of the viral genome and the formation of the 12 base-pair single-stranded "sticky" ends of mature lambda DNA. We have previously reported a kinetic analysis of the endonuclease activity of lambda terminase which showed an enzyme concentration-dependent change in the kinetic time course of the reaction [Tomka, M. A., & Catalano, C. E. (1993b) J. Biol. Chem. 268, 3056-3065]. We presented a model which suggested that the rate-limiting step in the nuclease reaction was the assembly of a catalytically competent prenicking complex. Here, we provide additional evidence for a slow assembly step in the nuclease reaction and demonstrate that the observed rate is affected by protein concentration, but not by the length of the DNA substrate. Consistent with our model, preincubation of terminase with DNA also yields an observable fast phase of the reaction, but only when large (> or = 3 kb) DNA substrates are used. Finally, we present data which demonstrate that phage lambda terminase can efficiently utilize DNA from the closely related phage phi21 as an endonuclease substrate and that the enzyme binds efficiently to the cosB region of both phage genomes. The implications of these results with respect to the assembly of a catalytically competent nucleoprotein complex required to initiate genome packaging are discussed.

Bacterial Proteins↗

PubMed1997

Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda.

A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome. We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA. Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding. Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.

Adenosine Triphosphate↗

PubMed1996

Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi.

Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.

Adenosine Triphosphatases↗

PubMed1995

Inactivation of DNA polymerase alpha-primase by acrolein: loss of activity depends on the DNA substrate.

We have utilized acrolein as a model compound to examine the biochemical behavior of chemically-modified DNA polymerase alpha-primase complex (pol alpha). We have found that acrolein irreversibly inactivates the DNA synthetic capacity of pol alpha polymerase in a time- and concentration-dependent manner. Double-stranded DNA protects pol alpha polymerase from inactivation when present during acrolein exposure, but single-stranded DNA, dATP and ATP do not. Strikingly, the activity of pol alpha polymerase is strongly dependent upon the DNA substrate utilized to assay catalytic activity after exposure to the aldehyde. The primase activity of pol alpha is also inactivated by exposure to acrolein, but the observed rate of inactivation is slower than that seen for DNA synthesis. Competitive labeling studies with [14C] iodoacetamide suggest that acrolein inactivation of the enzyme is mediated through the modification of protein sulfhydryl groups.

Acrolein↗

PubMed1995

Role of gpFI protein in DNA packaging by bacteriophage lambda.

One of the final steps in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of this monomer into a preformed capsid. Terminase enzymes are common to all of the double-stranded DNA phages, and in lambda this enzyme is responsible for both excision of a genome monomer from the concatemer and its insertion into the pro-capsid. We have previously demonstrated that the endonuclease activity of lambda terminase (cos-cleavage) was stoichiometric with enzyme and postulated that this was due to formation of a stable, postcleavage enzyme.DNA intermediate (complex I) (Tomka & Catalano, 1993b). Bacteriophage lambda gpFI protein is required for efficient assembly of the virus, and current models suggest that this protein increases the rate of pro-capsid binding to complex I. We show here that gpFI markedly stimulates cos-cleavage by lambda terminase, even in the absence of viral pro-capsids. Importantly, the observed increase in nicking activity did not result from an increase in the rate of cos-cleavage, but rather by an increase in turnover by the enzyme. These data suggest that gpFI destabilizes complex I, thus allowing terminase release from cos and catalytic turnover by the enzyme. The implications of these results with respect to terminase assembly onto viral DNA, nicking of the duplex, and subsequent translocation during packaging are discussed.

Bacteriophage lambda↗

PubMed1995

Virus DNA packaging: the strategy used by phage lambda.

Phage lambda, like a number of other large DNA bacteriophages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN to complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endonuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNu1, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

Amino Acid Sequence↗

PubMed1993

Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage lambda.

Terminases are enzymes common to all of the complex double-stranded DNA viruses and are required for viral assembly. These enzymes function to excise a single viral genome from a concatemeric DNA precursor and package it into a preformed protective protein shell or capsid. ATP hydrolysis by these enzymes has been described and appears to be critical to the packaging process. We have previously characterized the endonuclease activity of purified terminase from bacteriophage lambda [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065], and we describe here a kinetic characterization of the ATPase activity of the enzyme. lambda Terminase possesses a DNA-stimulated ATPase activity and hydrolyzes ATP to ADP and Pi. This activity requires divalent metal and is supported by all of the group IIa metals examined, as well as Mn2+. The reaction is also stimulated by NaCl, GTP, and dGTP. Of note is that neither of the guanosine nucleotides is hydrolyzed by the enzyme, while dATP is hydrolyzed at a rate comparable to that of ATP. Kinetic analysis of the ATPase activity revealed two apparent binding sites for ATP hydrolysis. The high-affinity site (Km = 5 microM) and low-affinity site (Km approximately 1.3 mM) hydrolyze ATP with kcat = 3 and 16 min-1, respectively. While the high-affinity site is unaffected by the presence of DNA, ATP hydrolysis at the low-affinity site is stimulated by DNA, which results from both a decrease in the Km and a concomitant increase in the kcat of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗

PubMed1993

Physical and kinetic characterization of the DNA packaging enzyme from bacteriophage lambda.

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of the viral genome into a preformed capsid. The overexpression of bacteriophage lambda-terminase in Escherichia coli has been previously reported (Chow, S., Daub, E., and Murialdo, H. (1987) Gene (Amst.) 60, 277-289), and we present here a purification scheme for the isolation of milligram quantities of protein which is homogenous ( > 97%) as determined by SDS-polyacrylamide gel electrophoresis. lambda-Terminase is composed of the gene products of Nu1 and A. Using N-terminal amino acid sequence analysis of the purified protein, we have determined a subunit stoichiometry of 2 gpNu1 polypeptides/gpA molecule in terminase holoenzyme. The circular dichroism spectrum for the purified holoenzyme has been obtained and is consistent with a protein complex composed primarily of alpha-helical structure. The endonucleolytic activity of the enzyme (the TER reaction) has been optimized with respect to pH, salt, and polyamine concentrations. Divalent metal ion is strictly required for the reaction and may be satisfied by either magnesium or manganese, but not by any of the other metals examined. E. coli integration host factor in amounts stoichiometric with the DNA substrate stimulates the TER reaction, but only when the enzyme is present in limiting amounts. Increasing the enzyme/DNA ratio attenuates the observed stimulation by integration host factor. A kinetic analysis of the TER reaction suggests that the assembly of multiple terminase promoters is required for efficient cleavage of viral DNA and that this reaction appears to be stoichiometric, rather than catalytic under the reaction conditions utilized. The implications of these results with respect to the packaging of viral DNA by terminase enzymes are discussed.

Bacterial Proteins↗

PubMed1990

Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts.

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.

Amino Acid Sequence↗

PubMed1989

Reactions of the protein radical in peroxide-treated myoglobin. Formation of a heme-protein cross-link.

Reaction of horse myoglobin with H2O2 oxidizes the iron to the ferryl (Fe(IV) = O) state and produces a protein radical that is rapidly dissipated by poorly understood mechanisms. As reported here, the reaction with H2O2 results in covalent binding of up to 18% of the prosthetic heme group to the protein. The chromophore of the protein-bound prosthetic group is very similar to that of heme itself. High performance liquid chromatography of tryptic digests indicates that the formation of heme-bound peptides is associated with disappearance of the peptide with the sequence YLE-FISDAIIHVLHSK corresponding to residues 103-118 of horse myoglobin. Amino acid analysis, terminal amino acid sequencing, and liquid secondary ion mass spectrometry establish that the heme is primarily attached to this peptide. The heme appears to be bound to the tyrosine residue because the tyrosine is the only amino acid that disappears from the amino acid analysis. The mass spectrometric data indicates that the heme-peptide is formed without addition or loss of an oxygen or other major structural fragment. The site of attachment to the heme group has not been unambiguously determined, but the heme vinyl groups are not essential for the reaction because equal cross-linking is observed in H2O2-treated mesoheme-reconstituted myoglobin. The results are most consistent with binding of tyrosine 103 to a meso-carbon of the prosthetic heme group.

Amino Acids↗

PubMed1989

Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor.

The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.

Adenosine Triphosphate↗

PubMed1987

Oxene transfer, electron abstraction, and cooxidation in the epoxidation of stilbene and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by hemoglobin.

Hemoglobin plus H2O2 oxidizes trans-stilbene to trans-stilbene oxide, cis-stilbene to cis- and trans-stilbene oxide, and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to anti-trans-7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Oxidation of cis- and trans-stilbene to the corresponding cis- and trans-epoxides proceeds exclusively with incorporation of oxygen from the peroxide. Oxidation of cis-stilbene to the trans-epoxide, however, proceeds without detectable incorporation of oxygen from the peroxide and partial incorporation of oxygen from O2. The epoxidations in which stereochemistry is conserved thus appear to involve ferryl oxygen transfer, whereas the epoxidations in which stereochemistry is inverted are proposed to involve protein-mediated cooxidation [Ortiz de Montellano, P.R., & Catalano, C.E. (1985) J. Biol. Chem. 260, 9265-9271] and possibly electron abstraction-water addition. The epoxidation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene incorporates oxygen from H2O2 and H2O but not O2. The oxidation of this substrate is thus consistent with ferryl oxygen transfer and electron abstraction but not protein-mediated cooxidation.

Animals↗

PubMed1987

Structure-mechanism relationships in hemoproteins. Oxygenations catalyzed by chloroperoxidase and horseradish peroxidase.

Chloroperoxidase and H2O2 oxidize styrene to styrene oxide and phenylacetaldehyde but not benzaldehyde. The epoxide oxygen is shown by studies with H2(18)O2 to derive quantitatively from the peroxide. The epoxidation of trans-[1-2H]styrene by chloroperoxidase proceeds without detectable loss of stereochemistry, as does the epoxidation of styrene by rat liver cytochrome P-450, although much more phenylacetaldehyde is produced by chloroperoxidase than cytochrome P-450. Chloroperoxidase and cytochrome P-450 thus oxidize styrene by closely related oxygen-transfer mechanisms. Horseradish peroxidase does not oxidize styrene but does oxidize 2,4,6-trimethylphenol to 2,6-dimethyl-4-hydroxymethylphenol. The new hydroxyl group is partially labeled in incubations with H2(18)O but not H2(18)O2. The hydroxyl group thus appears to be introduced by addition of oxygen to the benzylic radical and water to the quinone methide intermediate but not by a cytochrome P-450-like oxene transfer mechanism. The results support the thesis that substrates primarily or exclusively react with the heme edge of horseradish peroxidase but are able to react with the ferryl oxygen of chloroperoxidase.

Acetaldehyde↗

PubMed1985

Epoxidation of styrene by hemoglobin and myoglobin. Transfer of oxidizing equivalents to the protein surface.

Methemoglobin and metmyoglobin catalyze the H2O2-dependent oxidation of styrene to styrene oxide and benzaldehyde. The formation of styrene oxide requires molecular oxygen as well as H2O2 but does not, as shown by inhibitor studies, involve the superoxide or hydroxyl radicals. Approximately 38, 67, and 78% of the oxygen in styrene oxide derives from 18O2 in the reactions catalyzed, respectively, by bovine hemoglobin, sperm whale myoglobin, and equine heart myoglobin, whereas 70, 55, and 35% of the oxygen can be shown to be derived from [18O]H2O2. However, a larger fraction of the epoxide oxygen than suggested by the labeling data (perhaps all) derives from molecular oxygen rather than H2O2 because the hemoproteins produce molecular oxygen from the peroxide. The epoxidation of styrene by methemoglobin gives equal amounts of the R and S enantiomers and, as shown by studies with trans-[1-2H]styrene, proceeds with partial (33%) loss of the olefin stereochemistry. The results are rationalized by H2O2-dependent formation of a protein radical that combines with molecular oxygen to give a protein-peroxy radical that oxidizes styrene.

Animals↗