Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions.

Polarographic measurements showed that N₃^? and halides $\text{in}$ hibit the activity of bovine Cu, Zn superoxide dismutase in a competitive fashion, as previously demonstrated for CN^? and OH^?. All anions increase the spin-lattice nuclear magnetic relaxation time (T₁) of aqueous solutions of the enzyme as well, but the stability constants measured from T₁ data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the catalytic action at the water coordination position of the enzyme copper, and that these inhibitors may have a greater affinity for the cuprous form of the enzyme which is generated in the catalytic cycle.     

Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies.

We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism.     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56-61 and 99-104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.     

Ultrasonic study of the kinetics of counterion site binding in aqueous solutions of polyelectrolytes

The excess ultrasonic absorption due to counterion binding has been studied as a function of frequency for a series of polysalts in the range 1–150 MHz. All the relaxation spectra can be represented by a relaxation equation with two relaxation terms. The relaxation frequencies appear concentration independent and the relaxation amplitudes seem proportional to concentration. The low frequency relaxation process appears to depend mainly on the nature of the counterion while the high frequency relaxation process seems to be mostly dependent on the nature of the polyion. These results are quite similar to those obtained in ultrasonic studies of ion-pairing in solutions of divalent sulfates. The kinetic model used for the quantitative analysis of these results has been modified for polysalts through introducing the concept of“counterion condensation”. In this modified model the excess absorption is assigned to the perturbation by the ultrasonic waves of the equilibria between the three states of hydration of ths complex formed by a counterion and that part of the polyion where it is bound. Analytical expressions of the relaxation amplitudes have been derived using classical procedures for this modified kinetic model. In the case of cobalt-polyphosphate (Co-PP), the ultrasonic data together with the results of NMR measurements on either Co²⁺ or Co-PP have been used for the evaluation of the volume changes, the rate constants and the fractions of counterions in the three states of hydration involved in the binding equilibria. The volume changes obtained in this manner depend only slightly on the method of calculation and appear to be consistent with volume changes for outer-sphere and inner-sphere complex formation. These results are discussed.     

HNMR of succinate binding to aspartate transcarbamylase. A comparison of results in D2O and H2O.

The interaction of succinate with asparatete transcarbamylase from Escherichia coli has been studied by magnetic resonance relaxation measurements of the dicarboxylic acid methylene protons in H2O solutions. The pH and temperature dependence of the relaxation in the presence of either native asparte transcarbamylase or its catalytic subunit in H2O solutions is qualitatively very similar to the corresponding situation utilizing D2O as the solvent. From previous result of measurements in D2O[C.B. Beard and P.G. Schmidt, Biochemistry 12(1973)2255] a mechanism was proposed involving 2 protonated groups affecting succinate binding and titratable over the pH range 7-10. Quantitatively, fitting the data from H2O solutions to the mechanism yeilds values of the fitting parameters generally in good agreement with the D2O experiments. The main exceptions are the pKa values calculated for the two titratable groups. For these species the values obtained in the presence of the catalytic subunit are 6.7 and 7.8 in H2O solutions versus 7.3 and 8.6 in D2O solutions. In the presence of native enzyme the corresponding values are 6.8 and 8.3 in H2O versus 7.6 and 9.2 in D2O. These observed differences are consistent with differences in ionization constants of weak acids in D2O relative to H2O. The results imply that succinate interaction with the enzyme active site is similar in the two solvents.     

Characterisation of interchain association in polysaccharide solutions by ultrasonic relaxation and velocity

The amplitude of ultrasonic relaxation in aqueous solutions of disordered polysaccharides shows a marked increase with increasing degree of coil overlap and, at comparatively low concentrations, attains values comparable to those observed in polysaccharide gels. Mechanical spectroscopy studies indicate that, on the ultrasonic timescale, dynamic networks formed by polymer entanglement in solution are indistinguishable from true gels. In both cases the intense relaxations observed are attributed predominantly to motion of solvent within the polymer network. Due to the inherent stiffness of most polysaccharides, formation of a highly entangled network structure (with consequent enhancement of ultrasonic relaxation) occurs at much lower concentrations than for typical synthetic polymers. The onset of coil overlap (c¹ transition) is accompanied by an abrupt change in the concentration dependence of ultrasonic velocity. Results for the conformationally rigid polysaccharide xanthan, suggest that velocity measurements may offer a convenient method for determination of c¹ in systems where the normal viscometric characterisation is impossible.     

Intensification of biokinetics of enzymes using ultrasound-assisted methods: a critical review

The use of low intensity ultrasound has gotten surprising consideration over the last decade as a method for enhancing the catalytic activity of enzyme. Ultrasounds have the potential to significantly influence the activity of the enzymatic processes, provided that the energy input is not so high as to inactivate the enzyme. By providing the variation in parameters, various physical and chemical effects can be attained that can enhance the enzymatic reaction. Ultrasonic reactors are known for their application in bioprocesses. However, the potential of their applications is still limited broadly due to the lack of proper information about their operational and performance parameters. In this review, the detailed information about ultrasonic reactors is provided by defining the different types of reactors and number and position of ultrasonic transducers. Also, it includes mechanism of intensification and influence of ultrasonic parameters (intensity, duty cycle, and frequency) and enzymatic factors (enzyme concentration, temperature, and pH) on the catalytic activity of enzyme during ultrasound treatment.     

Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes.

Nuclear magnetic relaxation methods were used to investigate the interaction of the inhibitor succinate with aspartate transcarbamylase from Escherichia coli. Over the pH range 7 to 9, the dissociation constant for succinate remains less than the inhibitor concentration used for most of this work (0.05 M). As a result, the enzyme predominantly exists in a single "gross" conformational state. Succinate binding to this enzyme state (generally known as the R form) parallels the behavior seen previously with the isolated catalytic subunit (Beard, C. B., and Schmidt, P.G. (1973) Biochemistry 12, 2255-2264). The pH and temperature dependence of succinate proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl phosphate, is interpreted in terms of a binding mechanism involving three forms of the enzyme, differing by their states of protonation. The least protonated form of the enzyme does not interact with succinate, the singly protonated species binds succinate to form a rapidly dissociating complex, and the doubly protonated species undergoes a conformational isomerization upon succinate binding, yielding a slow exchange complex. Relaxation data provide sufficient information to determine pKa values of 7.2 and 8.9 for two ionizing groups, as well as the dissociation constant for succinate in the fast exchange complex, Kd =1.6 X 10(-2) M. Rate constants for the forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1, respectively, indicate a significantly slower reverse rate from that obtained in the earlier NMR study of the isolated catalytic subunit. In experiments where the succinate concentration was varied, the relaxation rates showed sigmoidal binding of that ligand to the fast exchange complex above pH 9.1, (a) indicating cooperative binding of succinate, and (b) suggesting that above pH 9.1, the system cannot be characterized by a single dissociation constant, ionization constant, or relaxation effect. CTP and ATP were tested for their ability to affect succinate binding to the fast exchange complex. Heterotropic interactions were observed for CTP but not for ATP. Addition of low concentrations of the transition state analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl phosphate-succinate complex sharply decreased the relaxation rate, indicating that the measurements are sensitive only to succinate bound specifically to the active site.     

Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes

Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH(*)) are quantified using (15)N nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8 T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290-320 K and at a static magnetic field strength of 14.1 T. Results for ttRNH(*) are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the picosecond-to-nanosecond time scale generally are similar for ttRNH(*) and ecRNH. beta-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH(*) than in ecRNH. Rigidity in the two beta-strands and the glycine-rich region, located along the periphery of the central beta-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH(*) than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of approximately 15 kJ/mol in the apparent activation energies for ttRNH(*) residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between alpha-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.     

Structure and dynamics of pin1 during catalysis by NMR

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.     

Study of the penicillin amidase from E. coli. An ultrasonic method of studying the ph- and temperature-conformational transitions in the active center of the enzyme]

pH and temperature conformation transitions in the active center of penicillin amidase i.e. penicillinamidohydrolase E.C. 3.5.I.II were investigated by means of the kinetic method and a new ultrasonic method. It was shown that the catalytic activity of the enzyme was controlled by 2 ionogenic groups with pK 6.1 and 10.2. The study of penicillinamidase by means of the ultrasonic method showed that the ionogenic group with pK 10 was responsible for maintaining the catalytically active conformation of the enzyme active center. Investigation of the temperature relation between the kinetic parameters of the enzymatic hydrolysis of benzylpenicillin catalyzed by penicillin amidase and the data on the effect of ultrasound on the enzyme showed that the enzyme was subjected to the temperature conformation transiton. The temperature and thermodynamic parameters of the conformation transition were determinded (T=318 degrees K, delta H=81 kcal/mole and delta S=255 e.u.). The structure of the active center of the enzyme is discussed on the basis of the data obtained.     

The p53 tumor suppressor stimulates the catalytic activity of human topoisomerase IIalpha by enhancing the rate of ATP hydrolysis

DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated approximately 2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha.     

Probing the flexibility of the DsbA oxidoreductase from Vibrio cholerae--a 15N - 1H heteronuclear NMR relaxation analysis of oxidized and reduced forms of DsbA

We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. The reduced structure shows a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helical domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerably more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike's information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the pico- to nanosecond and micro- to millisecond timescale. Many significant changes in dynamics are located either close to the active site or at the insertion points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect the catalytic cleft of the enzyme. Taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.     

Evidence for dynamically determined conformational states in rhodanese catalysis

Physical and kinetic studies have been used to explore hysteretic effects that are observed in rhodanese catalysis at pH 5 and also at neutral pH when the ionic strength of the medium is high. Experiments that involve observation of changes in intrinsic protein fluorescence of the enzyme and kinetic investigation of its interactions with product thiocyanate anion at pH 5 have implicated enzyme isomerization as the cause of hysteresis. Taken all together, the data indicate that the conformations of enzyme forms in the catalytic cycle are dynamically determined, depending on the relative rates of conformational relaxation and catalysis as influenced by the concentrations of substrates and products.     

Mechanism of phenylalanine regulation of phenylalanine hydroxylase

The mechanism of phenylalanine regulation of rat liver phenylalanine hydroxylase was studied. We show that phenylalanine "activates" phenylalanine hydroxylase, converting it from an inactive to active form, by binding at a true allosteric regulatory site. One phenylalanine molecule binds per enzyme subunit; it remains at this site during catalytic turnover and, while there, cannot be hydroxylated. Loss of phenylalanine from the site causes a loss of enzymatic activity. The rate of loss of activation is dramatically slowed by phenylalanine, which kinetically "traps" activated enzyme during relaxation from the activated to unactivated state. An empirical equation is presented which allows calculation of relaxation rates over a wide range of temperatures and phenylalanine concentrations. Kinetic trapping by phenylalanine is a novel effect. It was analyzed in detail, and its magnitude implied that phenylalanine activation involves cooperativity among all four subunits of the enzyme tetramer. A regulatory model is presented, accounting for the properties of the phenylalanine activation reaction in the forward and reverse directions and at equilibrium. Fluorescence quenching studies confirmed that activation increases the solvent accessibility of the enzyme's tryptophan residues. Physical and kinetic properties of purified phenylalanine hydroxylase from rat, rabbit, baboon, and goose liver were compared. All enzymes were remarkably alike in catalytic and regulatory properties, suggesting that control of this enzyme is similar in mammals and birds.     

Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.     

Interaction of T4 endonuclease V with DNA: importance of the flexible loop regions in protein-DNA interaction

T4 endonuclease V (T4 endo V), a thymine dimer-specific DNA repair enzyme, and its interaction with DNA were investigated by nuclear magnetic resonance (NMR) spectroscopy. Backbone resonance assignment, chemical shift mapping, and 15N relaxation measurements were employed to the free and DNA-bound enzymes. The secondary structure and the tertiary fold of T4 endo V in solution were consistent with those from the crystallographic study. The backbone 1H and 15N chemical shift perturbation upon the addition of DNA without a lesion revealed that the residues including Arg3, Arg22-Arg26, Lys45-Phe60, and Lys86-Thr88 participate in DNA binding. However, when DNA with a lesion was added to the enzyme and concomitantly the catalytic reaction was completed, the resonances of Arg22, Glu23, and Arg26, which constitute the catalytic active site, and the resonance of Thr88, were perturbed in a different manner. The region around Lys45-Ser47 was found to be involved in DNA binding, which have not been reported elsewhere. The backbone relaxation measurements of the free and DNA-bound enzymes indicated that two loop regions, Lys45-Phe60 and Lys86-Asp92, show the high degree of backbone flexibility. These results imply that two flexible loop regions may play an important role in DNA binding and in scanning along DNA duplex to search the thymine dimer sites in UV-damaged DNA.     

Kinetic and magnetic resonance studies of substrate binding to galactose oxidase copper(II)

Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate beta-O-methyl-D-galactopyranoside (beta-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN- or F- are used as inhibitors of GOase with beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O2, CN- and beta-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN- binds to the GOase Cu(II) at an equatorial coordination site, as does the F- detected in esr experiments. Thus, the results from the various competition experiments supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.     

Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km.

If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.     

Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis

Insertion and deletion mutagenesis within the gene topA of Escherichia coli encoding DNA topoisomerase I was carried out to test the existence of subdomains in the enzyme and the relationship between the slow-growth topA- phenotype and the known DNA relaxation activity of the enzyme. All mutants that show no detectable DNA relaxation activity in cell extracts fail to complement the temperature-sensitive growth defect of strain AS17 topAam harboring a plasmid-borne temperature-sensitive suppressor tRNA. All mutants that show partial or full levels of DNA relaxation activity in cell extracts (relative to activity in extracts of wild-type cells) can complement this defect. The carboxyl-proximal 25% of the enzyme appears to be in a domain that is dispensable both in terms of the catalytic function of the enzyme and its biological role. Analysis of the mutant enzyme also indicates that the formation of the covalent topoisomerase-DNA complex is correlated with the DNA relaxation activity, which supports the notion that the covalent complex is an obligatory intermediate in the catalysis of DNA topoisomerization.