Methane monooxygenase hydroxylase and B component interactions

The interaction of the soluble methane monooxygenase regulatory component (MMOB) and the active site-bearing hydroxylase component (MMOH) is investigated using spin and fluorescent probes. MMOB from Methylosinus trichosporium OB3b is devoid of cysteine. Consequently, site-directed mutagenesis was used to incorporate single cysteine residues, allowing specific placement of the probe molecules. Sixteen MMOB Cys mutants were prepared and labeled with the EPR spin probe 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (MSL). Spectral evaluation of probe mobility and accessibility to the hydrophilic spin-relaxing agent NiEDDA showed that both properties decrease dramatically for a subset of the spin labels as the complex with MMOH forms, thereby defining the likely interaction surface on MMOB. This surface contains MMOB residue T111 thought to play a role in substrate access into the MMOH active site. The surface also contains several hydrophilic residues and is ringed by charged residues. The surface of MMOB opposite the proposed binding surface is highly charged, consistent with solvent exposure. Probes of both of the disordered N- and C-terminal regions remain highly mobile and exposed to solvent in the MMOH complex. Spin-labeling studies show that residue A62 of MMOB is located in a position where it can be used to monitor MMOH-MMOB complex formation without perturbing the process. Accordingly, steady-state kinetic assays show that it can be changed to Cys (A62C) and labeled with the fluorescent probes 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) or 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) without loss of the ability of MMOB to promote turnover. The BADAN fluorescence is partially quenched and red shifted as the complex with MMOH forms, allowing affinity measurements. It is shown that the high affinity of labeled MMOB (K(D) = 13.5 nM at pH 6.6, 25 degrees C) for the oxidized MMOH decreases substantially with increasing pH and increasing ionic strength but is nearly unaffected by addition of nonionic detergents. Similarly, the fluorescence anisotropy of the 1,5-IAEDANS-labeled A62C-MMOH complex is perturbed by salts but not nonionic detergents. This suggests that the MMOB-MMOH complex is stabilized by electrostatic interactions consistent with the characteristics of the proposed binding surface. Reduction of MMOH results in a 2-3 order of magnitude decrease in the affinity of the BADAN-labeled A62C-MMOB-MMOH complex, consistent with previous indications of structural change associated with reduction of the active site dinuclear iron cluster. Utilizing BADAN-labeled MMOB, the association and dissociation rate constants for the MMOB-MMOH binding reaction were determined and found to be consistent with a two-step process, possibly involving rapid association followed by a slower conformational change. The latter may be related to the regulation of substrate access into the active site of MMOH.     

Methane monooxygenase: purification and properties of flavoprotein component

An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant (S20,W) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe2S2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.     

Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

The soluble methane monooxygenase (MMO) system, consisting of reductase, component B, and hydroxylase (MMOH), catalyzes NADH and O2-dependent monooxygenation of many hydrocarbons. MMOH contains 2 mu-(H or R)oxo-bridged dinuclear iron clusters thought to be the sites of catalysis. Although rapid NADH-coupled turnover requires all three protein components, three less complex systems are also functional: System I, NADH, O2, reductase, and MMOH; System II, H2O2 and oxidized MMOH; System III, MMOH reduced nonenzymatically by 2e- and then exposed to O2 (single turnover). All three systems give the same products, suggesting a common reactive oxygen species. However, the distribution of products observed for most substrates that are hydroxylated in more than one position is different for each system. For several of these substrates, addition of component B to Systems I, II, or III causes the product distributions to shift dramatically. These shifts result in identical product distributions for Systems I and III in which MMOH passes through the 2e- reduced state ([Fe(II).Fe(II)]) during catalysis. In contrast, System II (in which MMOH probably does not become reduced) generally gives a unique product distribution. It is proposed that changes in MMOH structure occurring upon diiron cluster reduction and/or component complex formation cause substrates to be presented differently to the activated oxygen species. Kinetic studies show that component B strongly activates System I and, in most cases, strongly deactivates System II. The effect of component B on product distribution of System I (and III) occurs at less than 5% of the MMOH concentration, while nearly stoichiometric concentrations are required to maximize the rate of System I. This shows that component B has at least two roles in catalysis. EPR monitored titration of reduced MMOH ([Fe(II).Fe(II)]) with component B suggests that the effect of substoichiometric component B on product distribution is due to hysteresis in the MMOH conformational changes.     

Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis

Abstract Methylosinus trichosporium OB3b synthesizes a soluble cytoplasmic methane monooxygenase when grown in copper-depleted medium and a membrane-bound particulate methane monooxygenase under copper-replete conditions. The genes encoding the hydroxylase component of soluble methane monooxygenase, carried on a plasmid in Escherichia coli , were insertionally inactivated using a kanamycin cassette and transferred back into M. trichosporium by conjugation. Marker-exchange mutagenesis, via a double homologous recombination event, yielded a soluble methane monooxygenase-negative mutant which grew only on methane using the particulate methane monooxygenase during copper-replete growth conditions, thus proving that the two methane oxidation systems in this methanotroph are genetically distinct.     

Identification of critical steps governing the two-component alkanesulfonate monooxygenase catalytic mechanism

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon-sulfur bond from sulfonated substrates. A mechanism involving acid-base catalysis has been proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from the alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO(-) intermediate. The pH profiles of k(cat) indicate that catalysis by SsuD requires a group with a pK(a) of 6.6 ± 0.2 to be deprotonated and a second group with a pK(a) of 9.5 ± 0.1 to be protonated. The upper pK(a) value was not present in the pH profiles of k(cat)/K(m). Several conserved amino acid residues (His228, His11, His333, Cys54, and Arg226) have been identified as having potential catalytic importance due to the similar spatial arrangements with close structural and functional relatives of SsuD. Substitutions to these amino acid residues were generated, and the pH dependencies were evaluated and compared to wild-type SsuD. Although a histidine residue was previously proposed to be the active site base, the His variants possessed similar steady-state kinetic parameters as wild-type SsuD. Interestingly, R226A and R226K SsuD variants possessed undetectable activity, and there was no detectable formation of the C4a-(hydro)peroxyflavin intermediate for the Arg226 SsuD variants. Guanidinium rescue with the R226A SsuD variant resulted in the recovery of 1.5% of the wild-type SsuD k(cat) value. These results implicate Arg226 playing a critical role in catalysis and provide essential insights into the mechanistic steps that guide the SsuD desulfonation process.     

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase

Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the OH-->EH and PM-->F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.     

Oxygen activation catalyzed by methane monooxygenase hydroxylase component: proton delivery during the O-O bond cleavage steps

The effects of solvent pH and deuteration on the transient kinetics of the key intermediates of the dioxygen activation process catalyzed by the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been studied. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains a carboxylate- and oxygen-bridged binuclear iron cluster that catalyzes O2 activation and insertion chemistry. The diferrous MMOH-MMOB complex reacts with O2 to form a diferrous intermediate compound O (O) and subsequently a diferric intermediate compound P (P), presumed to be a peroxy adduct. The O decay reaction was found to be pH-independent within error at 4 degrees C (kobs = 22 +/- 2 s-1 at pH 7.7; kobs = 26 +/- 2 s-1 at pH 7.0). In contrast, the P formation rate was found to decrease sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kP = 9.1 +/- 0.9 s-1 achieved near pH 6.5. The formation of P was slower than the disappearance of O, indicating that at least one other undetected intermediate (P) must form in between. P decays spontaneously to the highly chromophoric intermediate, compound Q (Q). The decay rate of P matched the formation rate of Q, and both rates decreased sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kQ = 2.6 +/- 0.1 s-1 achieved near pH 6.5. No pH dependence was observed for the decay of Q. The formation and decay rates of P and the formation rate of Q decreased linearly with mole fraction of D2O in the reaction mixture. Kinetic solvent isotope effect values of kH/kD = 1.3 +/- 0.1 (P formation) and kH/kD = 1.4 +/- 0.1 (P decay and Q formation) were observed at 5 degrees C. The linearity of the proton inventory plots suggests that only a single proton is transferred in the transition state of the formation reaction for each intermediate. If these protons are transferred to the bound oxygen molecule, as formally required by the reaction stoichiometry, the data are consistent with a model in which water is formed concurrently with the formation of the reactive bis mu-oxo-binuclear Fe(IV) species, Q.     

Identification and characterization of the product release steps within the catalytic cycle of protochlorophyllide oxidoreductase

The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP(+) cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP(+) from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.     

Cytochrome c oxidase: charge translocation coupled to single-electron partial steps of the catalytic cycle

The paper presents a survey of time-resolved studies of charge translocation by cytochrome c oxidase coupled to transfer of the 1st, 2nd 3rd and 4th electrons in the catalytic cycle. Single-electron photoreduction experiments carried out with the A-class cytochrome c oxidases of aa(3) type from mitochondria, Rhodobacter sphaeroides and Paracoccus denitrificans as well as with the ba(3)-type oxidase from Thermus thermophilus indicate that the protonmotive mechanisms, although similar, may not be identical for different partial steps in the same enzyme species, as well as for the same single-electron transition in different oxidases. The pattern of charge translocation coupled to transfer of a single electron in the A-class oxidases confirms major predictions of the original model of proton pumping by cytochrome oxidase [Artzatbanov, V. Y., Konstantinov, A. A. and Skulachev, V.P. "Involvement of Intramitochondrial Protons in Redox Reactions of Cytochrome a." FEBS Lett. 87: 180-185]. The intermediates and partial electrogenic steps observed in the single-electron photoreduction experiments may be very different from those observed during oxidation of the fully reduced oxidase by O(2) in the "flow-flash" studies. .     

The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP.Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg(2+).PP(i) complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9A resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasma gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T.gondii).     

Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B

The regulatory component MMOB of soluble methane monooxygenase (sMMO) has been hypothesized to control access of substrates into the active site of the hydroxylase component (MMOH) through formation of a size specific channel or region of increased structural flexibility tuned to methane and O(2). Accordingly, a decrease in the size of four MMOB residues (N107G/S109A/S110A/T111A, the Quad mutant) was shown to accelerate the reaction of substrates larger than methane with the reactive MMOH intermediate Q [Wallar, B. J., and Lipscomb, J. D. (2001) Biochemistry 40, 2220-2233]. Here, this hypothesis is tested by construction of single and double mutations involving the residues of the Quad mutant. It is shown that mutations of residues that extend into the core structure of MMOB alter many aspects of the MMOH catalyzed reaction but do not mimic the effects of the Quad mutant. In contrast, the MMOB residues that are thought to form part of the interface in the MMOH-MMOB complex increase active site accessibility as observed for the Quad mutant. In particular, the mutant T111A mimics most of the effects of the Quad mutant; thus, Thr111 is proposed to most directly control access. Unexpectedly, mutation of Thr111 to the larger Tyr greatly increases the rate constant for the reaction of larger substrates such as ethane, furan, and nitrobenzene with Q while decreasing the rate constant for the reaction with methane. Other steps in the cycle are dramatically slowed, the regiospecificity for nitrobenzene oxidation is altered, and 10-fold more T111Y than wild-type MMOB is required to maximize the rate of turnover. Thus, T111Y appears to make a more extensive change in local interface structure that allows hydrocarbons at least as large as ethane to bind and react with Q similarly. As a result, the bond cleavage rates for methane, ethane, and their deuterated analogues are shown for the first time to correlate with bond strength in accord with a mechanism in which C-H bond cleavage occurs during reaction of substrates with Q.     

Inhibition of pig liver esterase by trifluoromethyl ketones: modulators of the catalytic reaction alter inhibition kinetics

The kinetics of substrate hydrolysis by pig liver esterase show activation by various substrates as well as activation by organic solvents (both Vmax and Km increase) [Barker, D.L., & Jencks, W.P. (1969) Biochemistry 8, 3890]. The trifluoromethyl ketones 1,1,1-trifluoro-4-phenylbutan-2-one (TPB) and 1,1,1-trifluoro-4-(p-hydroxyphenyl)butan-2-one (OH-TPB) are slow, tight binding inhibitors of pig liver esterase with Ki values of 6.8 X 10(-9) M and 6.0 X 10(-9) M, respectively. Acetonitrile, TPB, and OH-TPB as well as the substrates pNPA and ethyl lactate caused a 15-130-fold increase in the rate of association (kon), and dissociation (koff), of the enzyme--TPB complex. The value of Ki (koff/kon) did not change. The effect cannot be attributed to half-sites reactivity since an increase in koff of OH-TPB is also observed with enzyme monomers. The results are consistent with a model proposed for the catalytic reaction (Barker & Jencks, 1969) which invokes two binding sites on each esterase subunit, a catalytic site and an effector site. Occupation of the effector site can increase koff and kon for the inhibitors TPB and OH-TPB. Not all compounds which bind at the effector site increase koff. Butanol binds at the effector site but does not effect koff of TPB. The results also indicate that an aromatic or a hydrophobic structure and a carbonyl group are required for optimal interaction with the effector site.     

The kinetics of ammonia release during the catalytic cycle of pig plasma amine oxidase

The time course of NH3 release during the catalytic cycle of pig plasma amine oxidase was followed by using the quenched-flow technique in conjunction with a sensitive assay for NH3. These studies were made under both air and O2-saturating conditions. The results establish unequivocally that NH3 is released in the step whereby a reduced enzyme species is re-oxidized by molecular O2 rather than in the step leading to the reduced enzyme. It is concluded that the catalytic cycle of the enzyme conforms to an aminotransferase mechanism rather than one in which an imine is an intermediate.     

Plasma ammonia concentrations and the slow component of oxygen uptake kinetics during cycle ergometry

The purposes of this study were to 1) compare the patterns of responses for plasma ammonia concentration ([NH3]) during moderate- vs. heavy-intensity cycle ergometry, and 2) examine the relationship between the V O2 slow component (V O 2SC) and plasma [NH3]. Thirteen healthy, untrained men (mean +/- SEM age = 24.8 +/- 0.6 years) performed a total of eight constant power output exercises (7 minutes in duration) at two different intensities (moderate, 60% gas exchange threshold [GET] = 60% of the gas exchange threshold; and heavy, Delta 50% = 50% of the difference between GET and V O2 max). Blood was collected from an antecubital vein before the exercise, during the last 3 minutes of the 6-minute warm-up, and during each minute of the 7-minute constant power output workbout. The time course of changes in plasma [NH3] and V O2 during the two constant power output exercise intensities were assessed separately using 2 (intensity) x 7 (time) repeated-measures analyses of variance. For 60% GET, there were no significant differences in the mean normalized plasma [NH3] during the 7-minute workbout. For Delta 50%, there was a significant increase in the mean normalized plasma [NH3] during the 7-minute workbout. These findings suggest a potential relationship between exercise-induced hyperammonemia and the V O 2SC during heavy-intensity exercise.     

Solution structure of component B from methane monooxygenase derived through heteronuclear NMR and molecular modeling

Methane monooxygenase (MMO) is a nonheme iron-containing enzyme which consists of three protein components: a hydroxylase (MMOH), an NADH-linked reductase (MMOR), and a small "B" component (MMOB) which plays a regulatory role. Here, 1H, 13C, 15N heteronuclear 2D and 3D NMR spectroscopy has been used to derive the solution structure of the 138 amino acid MMOB protein in the monomer state. Pulse field gradient NMR self-diffusion measurements indicate predominant formation of dimers at 1 mM MMOB and monomers at or below 0.2 mM. MMOB is active as a monomer. Aggregate exchange broadening and limited solubility dictated that multidimensional heteronuclear NMR experiments had to be performed at a protein concentration of 0.2 mM. Using 1340 experimental constraints (1182 NOEs, 98 dihedrals, and 60 hydrogen bonding) within the well-folded part of the protein (residues 36-126), MMOB structural modeling produced a well-defined, compact alpha/beta fold which consists of three alpha-helices and six antiparallel beta-strands arranged in two domains: a betaalphabetabeta and a betaalphaalphabetabeta. Excluding the ill-defined N- and C-terminal segments (residues 1-35 and 127-138), RMS deviations are 1.1 A for backbone atoms and 1.6 A for all non-hydrogen atoms. Compared to the lower resolution NMR structure for the homologous protein P2 from the Pseudomonas sp. CF600 phenol hydroxylase system (RMSD = 2.48 A for backbone atoms) (Qian, H., Edlund, U., Powlowski, J., Shingler, V., and Sethson, I. (1997) Biochemistry, 36, 495-504), that of MMOB reveals a considerably more compact protein. In particular, MMOB lacks the large "doughnut" shaped cavity reported for the P2 protein. This difference may result from the limited number of long-range NOEs that were available for use in the modeling of the P2 structure. This NMR-derived structure of MMOB, therefore, presents the first high-resolution structure of a small protein effector of a nonheme oxygenase system.     

Laser excitation studies of the product release steps in the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase

The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of approximately 72 s(-1). At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.     

Crystal structures of a purple acid phosphatase,representing different steps of this enzyme's catalytic cycle

Background  

Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood.     

Inducibility of metallothionein throughout the cell cycle.

Synchronized Chinese hamster cells were induced with ZnCl2 at multiple stages of the cell cycle and labeled with [35S]cysteine, and the 35S-labeled proteins were isolated and separated into metallothionein and nonmetallothionein fractions. Metallothionein was found to be inducible in all stages of the cell cycle and in G1-arrested cells.     

Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.     

Regulation of DNA repair throughout the cell cycle

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.