Ribonucleotide reductase from Escherichia coli. Identification of allosteric effector sites by chromatography on immobilized effectors.

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.     

Interaction of the recA protein of Escherichia coli with adenosine 5'-O-(3-thiotriphosphate)

Incubation of the recA protein of Escherichia coli with the ATP analog adenosine 5'-O-(3-thiotriphosphate) (ATP(gamma S)) in the presence of DNA produces an irreversible inhibition of ATPase activity, although in the presence of ATP, ATP(gamma S) shows an initial competitive inhibition. ATP(gamma S) is not appreciably hydrolyzed by recA protein and the inhibition of ATPase activity is due to the formation of stable complexes which contain equimolar amounts of ATP(gamma S) and recA protein. Formation of stable complexes requires DNA, which is also stably bound to recA protein in the presence of ATP(gammaS), at a ratio of 5 to 10 nucleotides/recA protein monomer. The DNA requirement is satisfied by either single-or double-stranded DNA, and in the latter case, the pH dependence is comparable to that observed for ATP hydrolysis. Binding of ATP(gamma S) is inhibited by other nucleoside di- and triphosphates with efficiencies corresponding to their inhibitory effects on the ATPase activity of recA protein.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.     

Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein

The properties of the high-affinity single-stranded DNA (ssDNA) binding state of Escherichia coli recA protein have been studied. We find that all of the nucleoside triphosphates that are hydrolyzed by recA protein induce a high-affinity ssDNA binding state. The effect of ATP binding to recA protein was partially separated from the ATP hydrolytic event by substituting calcium chloride for magnesium chloride in the binding buffer. Under these conditions, the rate of ATP hydrolysis is greatly inhibited. ATP increases the affinity of recA protein for ssDNA in a concentration-dependent manner in the presence of both calcium and magnesium chloride with apparent Kd values of 375 and 500 microM ATP, respectively. Under nonhydrolytic conditions, the molar ratio of ATP to ADP has an effect on the recA protein ssDNA binding affinity. Over an ATP/ADP molar ratio of 2-3, the affinity of recA protein for ssDNA shifts cooperatively from a low-to a high-affinity state.     

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.     

Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells.

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.     

Interaction of recA protein with a photoaffinity analogue of ATP, 8-azido-ATP: determination of nucleotide cofactor binding parameters and of the relationship between ATP binding and ATP hydrolysis

The binding and cross-linking of the ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (azido-ATP) with recA protein have been investigated, and through cross-linking inhibition studies, the binding of other nucleotide cofactors to recA protein has also been studied. The azido-ATP molecule was shown to be a good ATP analogue with regard to recA protein binding and enzymatic function by three criteria: first, the cross-linking follows a simple hyperbolic binding curve with a Kd of 4 microM and a cross-linking efficiency ranging from 10% to 70% depending on conditions; second, ATP, dATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) specifically inhibit the cross-linking of azido-ATP to recA protein; third, azido-ATP is a substrate for recA protein ATPase activity. Quantitative analysis of the cross-linking inhibition studies using a variety of nucleotide cofactors as competitors has shown that the binding affinity of adenine-containing nucleotides for recA protein decreases in the following order: ATP-gamma-S greater than dATP greater than ATP greater than adenylyl beta,gamma-imidodiphosphate (AMP-PNP) much greater than adenylyl beta,gamma-methylenediphosphate (AMP-PCP) approximately adenine. Similar competition studies also showed that nearly all of the other nucleotide triphosphates also bind to recA protein, with the affinity decreasing in the following order: UTP greater than GTP approximately equal to dCTP greater than dGTP greater than CTP. In addition, studies performed in the presence of single-stranded DNA demonstrated that the affinity of ATP, dATP, ATP-gamma-S, and AMP-PNP for recA protein is significantly increased. These results are discussed in terms of the reciprocal effects that nucleotide cofactors have on the modulation of recA protein--single-stranded DNA binding affinity and vice versa. In addition, it is demonstrated that nucleotide and DNA binding are necessary though not sufficient conditions for ATPase activity. The significance of this result in terms of the possible requirement of critically sized clusters of 15 or more recA protein molecules contiguously bound to DNA for ATPase activity is discussed.     

Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor

Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide and nucleoside triphosphate. The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) satisfies the NTP requirement. We show here that despite its activity in repressor cleavage, ATP gamma S is hydrolyzed at a negligible rate by the recA protein DNA-dependent nucleoside triphosphatase activity. In the presence of DNA, ATP gamma S binds tightly to recA protein in a complex that can be detected because it is trapped by a nitrocellulose filter. One ATP gamma S molecule is bound per recA monomer. These results suggest that a ternary complex of recA protein, DNA, and nucleoside triphosphate is the species active in repressor cleavage. The activation of recA protein by small, defined oligonucleotides in place of DNA is described and characterized.     

Identification of the dITP- and XTP-hydrolyzing protein from Escherichia coli

A hypothetical 21.0 kDa protein (ORF O197) from Escherichia coli K-12 was cloned, purified, and characterized. The protein sequence of ORF O197 (termed EcO197) shares a 33.5% identity with that of a novel NTPase from Methanococcus jannaschii. The EcO197 protein was purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration column. It hydrolyzed nucleoside triphosphates with an O6 atom-containing purine base to nucleoside monophosphate and pyrophosphate. The EcO197 protein had a strong preference for deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), while it had little activity in the standard nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP). These aberrant nucleotides can be produced by oxidative deamination from purine nucleotides in cells; they are potentially mutagenic. The mutation protection mechanisms are caused by the incorporation into DNA of unwelcome nucleotides that are formed spontaneously. The EcO197 protein may function to eliminate specifically damaged purine nucleotide that contains the 6-keto group. This protein appears to be the first eubacterial dITP- and XTPhydrolyzing enzyme that has been identified.     

Binding of RecA protein to single-stranded nucleic acids: spectroscopic studies using fluorescent polynucleotides

Binding of the recA gene product from Escherichia coli to single-stranded polynucleotides has been investigated using poly(dA) that have been modified by chloroacetaldehyde to yield fluorescent 1,N6-ethenoadenine (epsilon A) bases. A strong enhancement of the fluorescent quantum yield of poly(d epsilon A) is induced upon RecA protein binding. A 4-fold increase is observed in the absence of ATP or ATP gamma S and a 7-fold increase in the presence of either nucleoside triphosphate. RecA protein can bind to poly(d epsilon A) in the absence of both Mg2+ ions and ATP (or ATP gamma S) but Mg2+ ions are required to observe RecA protein binding in the presence of ATP (or ATP gamma S) at pH 7.5. ATP binding to the RecA-poly(d epsilon A) complex induces a dissociation of RecA from the polynucleotide followed by re-binding of [RecA-ATP-Mg2+] ternary complex. Whereas ATP-induced dissociation of RecA-poly(d epsilon A) complexes is a fast process, the subsequent binding reaction of [RecA-ATP-Mg2+] is slow. A model is proposed whereby [RecA-ATP-Mg2+] binding to poly(d epsilon A) involves slow nucleation and elongation processes along the polynucleotide backbone. The nucleation reaction is shown to involve at least a trimer or a tetramer. Polymerization of the [RecA-ATP-Mg2+] ternary complex stops when the polynucleotide is entirely covered with 6 +/- 1 nucleotides per RecA monomer. ATP hydrolysis then induces a release of RecA-ADP complexes from the polynucleotide template.     

Interaction of the recA protein of Escherichia coli with single-stranded DNA

The interaction of recA protein with single-stranded (ss) phi X174 DNA has been examined by means of a nuclease protection assay. The stoichiometry of protection was found to be 1 recA monomer/approximately 4 nucleotides of ssDNA both in the absence of a nucleotide cofactor and in the presence of ATP. In contrast, in the presence of adenosine 5'-O-(thiotriphosphate) (ATP gamma S) the stoichiometry was 1 recA monomer/approximately 8 nucleotides. No protection was seen with ADP. In the absence of a nucleotide cofactor, the binding of recA protein to ssDNA was quite stable as judged by equilibration with a challenge DNA (t1/2 approximately 30 min). Addition of ATP stimulated this transfer (t1/2 approximately 3 min) as did ADP (t1/2 approximately 0.2 min). ATP gamma S greatly reduced the rate of equilibration (t1/2 greater than 12 h). Direct visualization of recA X ssDNA complexes at subsaturating recA protein concentrations using electron microscopy revealed individual ssDNA molecules partially covered with recA protein which were converted to highly condensed networks upon addition of ATP gamma S. These results have led to a general model for the interaction of recA protein with ssDNA.     

Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.     

Effect of ligand binding on the tryptic digestion pattern of rat brain hexokinase: relationship of ligand-induced conformational changes to catalytic and regulatory functions

The Type I isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is comprised of N- and C-terminal domains, associated with regulatory and catalytic functions, respectively. Extensive sequence similarity between the domains is consistent with evolution of the enzyme by gene duplication and fusion. Cleavage at tryptic sites located in the C-terminal domain is markedly sensitive to ligands present during digestion, while analogous sites in the N-terminal domain are either resistant to trypsin or unaffected by the presence of ligands. These results imply a lack of structural equivalence between the N- and C-terminal domains, with the overall structure of the N-terminal domain being "tighter" and with a major component of ligand-induced conformational changes being focused in the C-terminal domain. Based on a previously proposed structure for brain hexokinase, protection by substrate hexoses is attributed to substrate-induced closing of a cleft in the C-terminal domain. Similar protection at C-terminal cleavage sites results from binding of inhibitory hexose-6-phosphates to the N-terminal domain. In addition, hexose-6-phosphates evoke cleavage at a site, T5, located in a region that has been associated with binding of ATP to the C-terminal domain. Thus, alterations in this region, coupled with reduced accessibility resulting from cleft closure, may account for the mutually exclusive binding of inhibitory hexose-6-phosphates and substrate ATP. In the absence of Mg2+, all nucleoside triphosphates examined (ATP, UTP, CTP, and GTP) protected against digestion by trypsin. In contrast, ATP-Mg2+ stabilized the C-terminal domain but destabilized the N-terminal domain, while the chelated forms of the other nucleoside triphosphates were similar to the unchelated forms in their effect on proteolysis; the unique response to ATP-Mg2+ reflects the specificity for ATP as a substrate.     

ATP-independent renaturation of complementary DNA strands by the mutant recA1 protein from Escherichia coli

In an effort to clarify the requirement for ATP in the recA protein-promoted renaturation of complementary DNA strands, we have analyzed the mutant recA1 protein which lacks single-stranded DNA-dependent ATPase activity at pH 7.5. Like the wild type, the recA1 protein binds to single-stranded DNA with a stoichiometry of one monomer per approximately four nucleotides. However, unlike the wild type, the mutant protein is dissociated from single-stranded DNA in the presence of ATP or ADP. The ATP analogue adenosine 5'-O-3' (thiotriphosphate) appears to stabilize the binding of recA1 protein to single-stranded DNA but does not elicit the stoichiometry of 1 monomer/8 nucleotides or the formation of highly condensed protein-DNA networks that are characteristic of the wild type recA protein in the presence of this analogue. The recA1 protein does not catalyze DNA renaturation in the presence of ATP, consistent with the dissociation of recA1 protein from single-stranded DNA under these conditions. However, it does promote a pattern of Mg2+-dependent renaturation identical to that found for wild type recA protein.     

Cooperative and allosterically controlled nucleotide binding regulates the DNA binding activity of NrdR

Ribonucleotide reductases (RNRs) are required for the synthesis of deoxyribonucleoside triphosphates (dNTPs) from ribonucleotides. In Escherichia coli, regulation of RNR expression is co‐ordinated with the cell cycle, and involves several regulatory proteins. One of these, NrdR, has recently been shown to regulate all three nrd operons that encode RNR isoenzymes. Repression by NrdR is believed to be stimulated by elevated dNTPs, although there is no direct evidence for this model. Here, we sought to elucidate the mechanism by which NrdR regulates nrd expression according to the abundance of (d)NTPs. We determined that ATP and dATP bind to NrdR in a negatively cooperative fashion, such that neither can fully occupy the protein. Both nucleotides also appear to act as positive heterotropic effectors, since the binding of one stimulates binding of the other. Nucleotide binding stimulates self‐association of NrdR, with tri‐ and diphosphates stimulating oligomerization more effectively than monophosphates. As‐prepared NrdR contains (deoxy)nucleoside monophosphates, diphosphates and triphosphates, and its DNA binding activity is inhibited by triphosphates and diphosphates but not by monophosphates. We propose a model in which NrdR selectively binds (deoxy)nucleoside triphosphates, which are hydrolysed to their monophosphate counterparts in order to regulate DNA binding.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.     

Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products

We report here results of crystallographic studies at 3.0 Å resolution of complexes of phosphate ligands with aspartate carbamoyltransferase from Escherichia coli. Specifically, we interpret the binding of CTP, ATP, 5-bromo-CTP, 8-bromo-GTP. formycin A 5′-triphosphate. 3,N⁶-etheno-ATP. phosphate/carbamoyl-d.l-aspartate and pyrophosphate to the catalytic and regulatory chains of the enzyme.We observed two modes of binding of ligands to the phosphate crevice of the catalytic chain. Pyrophosphate and phosphate penetrate deeply into the cleft between the two domains of a catalytic monomer. In contrast. ATP, CTP. formycin A 5′-triphosphate and 3,N⁶-etheno-ATP bind to an exposed region of this cleft through their β and γ phosphates. Although the β and γ phosphates of 8-bromo-GTP bind to the same region as do the non-brominated nucleotides. 8-bromo-GTP interacts with the protein through all three of its phosphates and its ribose.Ser52, Arg54. Thr55, Arg105, His134. Gln137 and Arg167 are residues of the catalytic chain near density corresponding to phosphate ligands. The interactions of phosphate ligands are consistent with results of nuclear magnetic resonance, kinetics and equilibrium binding studies.Nucleoside triphosphates also bind to the regulatory chain in two modes. ATP and CTP bind in similar conformations to nearly the same site of the allosteric domain. The effector 8-bromo-GTP interacts at a location that does not overlap with the ATP-CTP site. The phosphates are in an extended conformation for all effectors. Furthermore, ATP. 5-bromo-CTP and 8-bromo-GTP bind to the protein in the anti conformation.Interactions of ATP and CTP with the protein are essentially consistent with the proposals put forward by London &; Schmidt (1972). We suggest, however, a modification of the London &; Schmidt model on the basis of our results with 8-bromo-GTP. In addition, we propose that the allosteric binding sites of nucleoside triphosphates are coupled to each other through the N-terminal segments of monomers of a regulatory dimer.     

Nucleotide effects on kinetic properties of mitochondrial ATPase

The hydrolytic activity of mitochondrial ATPase, both in its soluble form as F1-ATPase, or as membrane bound in whole mitochondria, was affected by the presence of free nucleoside di- or triphosphates; these effects were largely depending not only on their concentration but also on the substrate concentration. The existence of a regulatory site or sites is proposed; these sites would have a higher affinity for the free nucleoside triphosphates than for the diphosphates, and the interaction of any of these nucleotides with the proposed regulatory site or sites would lead to an activation. The nucleotide regulatory site or sites seem to be different from the anion binding sites since neither free ATP nor free GTP compete with activating or inhibitory anions.     

Nucleoside triphosphate specificity of tubulin

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.