Catalytic function of the residues of phenylalanine and tyrosine conserved in squalene-hopene cyclases.

Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-pi interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three residues, Y372, Y474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.     

Catalytic Function of the Residues of Phenylalanine and Tyrosine Conserved in Squalene-Hopene Cyclases

Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-π interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three redsidues, Y372, Y474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.     

Evidence for a dynamic role for proline376 in the purine-cytosine permease of Saccharomyces cerevisiae.

The purine-cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem. 272, 9697-9702] suggest that the hydrophilic segment 371-377 (-I-A-N-N-I-P-N-) of the polypeptide chain may play a key role in the correct three-dimensional structure of the active carrier. This paper describes the effects of mutations in this particular segment: a four-residue deletion, Delta374-377, and two substitutions, P376G and P376R. The Delta374-377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild-type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base-binding sites [1000-1200 pmol.(mg protein)-1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5-6.0), showing that the conformational change induced by ligand binding was no longer present. Our results show that the 374-377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a beta-turn motif, which must play a crucial dynamic role in the translocation process.     

Two consecutive aspartic acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase

Acetohydroxy acid synthase (AHAS) catalyzes the first common step in the biosynthesis pathway of the branch chain amino acids in plants and microorganisms. A great deal of interest has been focused on AHAS since it was identified as the target of several classes of potent herbicides. In an effort to produce a mutant usable in the development of an herbicide-resistant transgenic plant, two consecutive aspartic acid residues, which are very likely positioned next to the enzyme-bound herbicide sulfonylurea as the homologous residues in AHAS from yeast, were selected for this study. Four single-point mutants and two double mutants were constructed, and designated D374A, D374E, D375A, D375E, D374A/D375A, and D374E/D375E. All mutants were active, but the D374A mutant exhibited substrate inhibition at high concentrations. The D374E mutant also evidenced a profound reduction with regard to catalytic efficiency. The mutation of D375A increased the K(m) value for pyruvate nearly 10-fold. In contrast, the D375E mutant reduced this value by more than 3-fold. The double mutants exhibited synergistic reduction in catalytic efficiencies. All mutants constructed in this study proved to be strongly resistant to the herbicide sulfonylurea Londax. The double mutants and the mutants with the D375 residue were also strongly cross-resistant to the herbicide triazolopyrimidine TP. However, only the D374A mutant proved to be strongly resistant to imidazolinone Cadre. The data presented here indicate that the two residues, D374 and D375, are located at a common binding site for the herbicides sulfonylurea and triazolopyrimidine. D375E may be a valuable mutant for the development of herbicide-resistant transgenic plants.     

Control of substrate specificity by a single active site residue of the KsgA methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.     

Identification of selectivity-determining residues in cytochrome P450 monooxygenases: a systematic analysis of the substrate recognition site 5

The large and diverse family of cytochrome P450 monooxygenases (CYPs) was systematically analyzed to identify selectivity- and specificity-determining residues in the substrate recognition site 5, which is located in close vicinity to the heme center. A positively charged heme-interacting residue was identified in the structures of 29 monooxygenases and in 97.7% of the 6379 CYP sequences investigated here. This heme-interacting residue restricts the conformation of the substrate recognition site 5 and is preferentially located at position 10 or 11 after the conserved ExxR motif (in 94.4% of the sequences), in 3.3% of the sequences at position 9 or 12. As a result, a classification by the position of the heme-interacting residue allows to predict residues that are closest to the heme center and restrict its accessibility. In 98.4% of all CYP sequences a preferentially hydrophobic residue is located at position 5 after the ExxR motif that is predicted to point close to the heme center. Replacing this residue by hydrophobic residues of different size has been shown to change substrate specificity and regioselectivity for CYPs of different superfamilies. Twenty-seven percent of all CYPs are predicted to contain a second selectivity-determining residue at position 9 after the ExxR motif that can be identified by the pattern EXXR-X(7)-{P}-x-P-[HKR].     

ATP binding site on the C-terminus of the vanilloid receptor

Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labelling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

Introgression of wheat chromosome 2D or 5D into tritordeum leads to free-threshing habit.

Hexaploid tritordeum is the amphiploid derived from the cross between the diploid wild barley Hordeum chilense and durum wheat. The non-free-threshing habit is a constraint to this species becoming a new crop. Three tritordeum lines (HT374, HT376, and HT382) showing the free-threshing habit were selected from crosses between tritordeum and bread wheat. All three lines were euploids, as revealed by mitotic chromosome counting. Genomic in situ hybridization analysis made it possible to distinguish differences among these lines. While the line HT382 carries only 10 chromosomes from H. chilense, the lines HT374 and HT376 have 12. These results suggest that HT382 is a double chromosome substitution line between H. chilense and the wheat D genome, while HT374 and HT376 each have one pair of H. chilense (Hch) chromosomes substituted by wheat D chromosomes. Molecular characterization revealed that HT382 is a 1D/(1Hch), 2D/(2Hch) chromosome substitution line, whereas HT374 and HT376 have 5D/(5Hch) substitutions. On the basis of previous knowledge, it seems that the absence of chromosome 2Hch or 5Hch is more important for producing the free-threshing habit than the presence of chromosome 2D or 5D, while chromosome 1Hch seems to be unrelated to the trait. These free-threshing tritordeum lines constitute an important advance in the tritordeum breeding program.     

Mutations in the Human Immunodeficiency Virus Type 1 Integrase D,D(35)E Motif Do Not Eliminate Provirus Formation

The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3′-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 10³- to 10⁴-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721–728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per μg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3′-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.     

Identification of the carbonic anhydrase II binding site in the Cl(-)/HCO(3)(-) anion exchanger AE1

The human Cl(-)/HCO(3)(-) anion exchanger (AE1) possesses a binding site within its 33 residue carboxyl-terminal region (Ct) for carbonic anhydrase II (CAII). The amino acid sequence comprising this CAII binding site was determined by peptide competition and by testing the ability of truncation and point mutants of the Ct sequence to bind CAII with a sensitive microtiter plate binding assay. A synthetic peptide consisting of the entire 33 residues of the Ct (residues 879-911) could compete with a GST fusion protein of the Ct (GST-Ct) for binding to immobilized CAII, while a peptide consisting of the last 16 residues (896-911) could not. A series of truncation mutants of the GST-Ct showed that the terminal 21 residues of AE1 were not required for binding CAII. Removal of four additional residues (887-890) from the Ct resulted in loss of CAII binding. Acidic residues in this region (D887ADD) were critical for binding since mutating this sequence in the GST-Ct to DAAA, AAAA, or NANN caused loss of CAII binding. A GST-Ct construct mutated to D887ANE, the homologous sequence in AE2, could bind CAII. AE2 is a widely expressed anion exchanger and has a homologous Ct region with 60% sequence identity to AE1. A GST fusion protein of the 33 residue Ct of AE2 could bind to CAII similarly to the Ct of AE1. Tethering of CAII to an acidic motif within the Ct of anion exchangers may be a general mechanism for promoting bicarbonate transport across cell membranes.     

Identification of residues involved in catalytic activity of the inverting glycosyl transferase WbbE from Salmonella enterica serovar borreze

Synthesis of the O:54 O antigen of Salmonella enterica is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycosyl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-terminal domain, domain A. Based on structural data from the GT2 representative SpsA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380-6385, 1999), this domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second coordinating a Mn(2+) ion. Site-directed replacement of Asp41 (D41A) of WbbE, the analogue of the first Asp residue of SpsA, revealed that this is not required for activity. WbbE possesses three Asp residues near the position analogous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D93 or D96 may coordinate the Mn(2+) ion. Our studies also identified a C-terminal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an ED(Y) motif. The Glu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to reside on a turn. Combined with the alignment of the motif with potential catalytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is the catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogeneous family.     

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.     

Three-dimensional view of the surface motif associated with the P-loop structure: cis and trans cases of convergent evolution

Here we identify the determinants of the nucleotide-binding ability associated with the P-loop-containing proteins, inferring their functional importance from their structural convergence to a unique three- dimensional (3D) motif. (1) A new surface 3D pattern is identified for the P-loop nucleotide-binding region, which is more selective than the corresponding sequence pattern; (2) the signature displays one residue that we propose is the determinant for the guanine-binding ability (the residues aligned to ras D119; this residue is known to be important only in the G-proteins, we extend the prediction to all the other P-loop- containing proteins); and (3) two cases of convergent evolution at the molecular level are highlighted in the analysis of the active site: the positive charge aligned to ras K117 and the arginine residues aligned to the GAP arginine finger.The analysis of the residues conserved on protein surfaces allows one to identify new functional or evolutionary relationships among protein structures that would not be detectable by conventional sequence or structure comparison methods.     

Diversity in the SH2 domain family phosphotyrosyl peptide binding site

Src homology 2 (SH2) domains are approximately 100 residue phosphotyrosyl peptide binding modules found in signalling proteins and are important targets for therapeutic intervention. The peptide binding site is evolutionarily well conserved, particularly at the two major binding pockets, pTyr and pTyr + 3. We present a computational analysis of diversity within the peptide binding region and discuss molecular recognition beyond the conventional binding motif, drawing attention to novel conserved ligand interaction sites which may be exploitable in ligand binding studies. The peptide binding site is defined by selecting crystal contacts and domains are clustered according to binding site residue similarity. Comparison with a classification based on experimental peptide screening reveals a high level of qualitative agreement, indicating that the method is able independently to generate functional information. A conservation scoring method reveals extensive patches of conservation in some groups not present across the whole family, challenging the notion that the domains recognise only a linear phosphopeptide sequence. Conservation difference maps determine group-dependent clusters of conserved residues that are not seen when considering a larger experimentally determined group. Many of these residues contact the peptide outside the pTyr to pTyr + 3 motif, challenging the conventional view that this motif is largely responsible for ligand recognition and discrimination.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.     

Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments

Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.     

Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.