Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, C-terminal Src kinase (Csk)

Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.     

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.     

Control of substrate specificity by active-site residues in nitrobenzene dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the alpha subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics.     

Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.     

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar K_m for the substrate based on product formation rates and whole-cell kinetics.     

Substrate specificity of phosphorylase kinase: effects of heparin and calcium

Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2+; the rate of syntide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the Vmax and Km for these substrates were affected diversely by heparin and Ca2+. Heparin stimulated phosphorylase kinase maximally at pH 6.5, whereas the effect of Ca2+ was optimal at a pH above 8. However, the stimulator-related substrate specificity could not be explained by the different pH values at which the effects of the stimulators were assessed. Nor did substrate-directed effects by heparin or Ca2+ apparently play a role. No indications were found for a stimulator-dependent specificity in the phosphorylation of sites in protein substrates of phosphorylase kinase (phosphorylase b, the alpha- and beta-subunits of phosphorylase kinase, or glycogen synthase). The diverse substrate specificity of the calcium- and heparin-dependent activities of phosphorylase kinase could be explained in two ways: either by the existence of separate calcium- and heparin-stimulated catalytic sites, or by just one catalytic site with two active conformations. The second possibility is favored by the observation that both calcium and heparin stimulated the isolated gamma-subunit (gamma X calmodulin complex) of phosphorylase kinase.     

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.     

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.     

Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel

P450BM-3 is an extensively studied P450 cytochrome that is naturally fused to a cytochrome P450 reductase domain. Crystal structures of the heme domain of this enzyme have previously generated many insights into features of P450 structure, substrate binding specificity, and conformational changes that occur on substrate binding. Although many P450s are inhibited by imidazole, this compound does not effectively inhibit P450BM-3. Omega-imidazolyl fatty acids have previously been found to be weak inhibitors of the enzyme and show some unusual cooperativity with the substrate lauric acid. We set out to improve the properties of these inhibitors by attaching the omega-imidazolyl fatty acid to the nitrogen of an amino acid group, a tactic that we used previously to increase the potency of substrates. The resulting inhibitors were significantly more potent than their parent compounds lacking the amino acid group. A crystal structure of one of the new inhibitors bound to the heme domain of P450BM-3 reveals that the mode of interaction of the amino acid group with the enzyme is different from that previously observed for acyl amino acid substrates. Further, required movements of residues in the active site to accommodate the imidazole group provide an explanation for the low affinity of imidazole itself. Finally, the previously observed cooperativity with lauric acid is explained by a surprisingly open substrate-access channel lined with hydrophobic residues that could potentially accommodate lauric acid in addition to the inhibitor itself.     

Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site

Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.     

Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions

Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca(2+) signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous mu-calpain isoform by a peptide library-based approach using the proteolytic core of mu-calpain (muI-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for muI-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transfer-based substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The mu-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of alpha-spectrin or the proteolytic sequence consensus compiled from substrate alignments.     

Molecular basis for specificity of the extracytoplasmic thioredoxin ResA

ResA, an extracytoplasmic thioredoxin from Bacillus subtilis, acts in cytochrome c maturation by reducing the disulfide bond present in apocytochromes prior to covalent attachment of heme. This reaction is (and has to be) specific, as broad substrate specificity would result in unproductive shortcircuiting with the general oxidizing thioredoxin(s) present in the same compartment. Using mutational analysis and subsequent biochemical and structural characterization of active site variants, we show that reduced ResA displays unusually low reactivity at neutral pH, consistent with the observed high pKa values>8 for both active site cysteines. Residue Glu80 is shown to play a key role in controlling the acid-base properties of the active site. A model in which substrate binding dramatically enhances the reactivity of the active site cysteines is proposed to account for the specificity of the protein. Such a substratemediated activation mechanism is likely to have wide relevance for extracytoplasmic thioredoxins.     

Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.     

Current and prospective applications of non-proteinogenic amino acids in profiling of proteases substrate specificity

Abstract Proteases recognize their endogenous substrates based largely on a sequence of proteinogenic amino acids that surrounds the cleavage site. Currently, several methods are available to determine protease substrate specificity based on approaches employing proteinogenic amino acids. The knowledge about the specificity of proteases can be significantly extended by application of structurally diverse families of non-proteinogenic amino acids. From a chemical point of view, this information may be used to design specific substrates, inhibitors, or activity-based probes, while biological functions of proteases, such as posttranslational modifications can also be investigated. In this review, we discuss current and prospective technologies for application of non-proteinogenic amino acids in protease substrate specificity profiling.     

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.     

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.     

Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging

As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.     

Plant aminoaldehyde dehydrogenases oxidize a wide range of nitrogenous heterocyclic aldehydes

The metabolic degradation of aldehydes is catalyzed by oxidoreductases from which aldehyde dehydrogenases (EC 1.2.1) comprise nonspecific or substrate-specific enzymes. The latter subset is represented, e.g., by NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs; EC 1.2.1.19) oxidizing a group of naturally occurring ω-aminoaldehydes including polyamine oxidation products. Recombinant isoenzymes from pea (PsAMADH1 and 2) and tomato (LeAMADH1 and 2) were subjected to kinetic measurements with synthetic aldehydes containing a nitrogenous heterocycle such as pyridinecarbaldehydes and their halogenated derivatives, (pyridinylmethylamino)-aldehydes, pyridinyl propanals and aldehydes derived from purine, 7-deazapurine and pyrimidine to characterize their substrate specificity and significance of the resulting data for in vivo reactions. The enzymatic production of the corresponding carboxylic acids was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Although the studied AMADHs are largely homologous and supposed to have a very similar active site architecture, significant differences were observed. LeAMADH1 displayed the broadest specificity oxidizing almost all compounds followed by PsAMADH2 and 1. In contrast, LeAMADH2 accepted only a few compounds as substrates. Pyridinyl propanals were converted by all isoenzymes, usually better than pyridinecarbaldehydes and aldehydes with fused rings. The K (m) values for the best substrates were in the range of 10(-5)-10(-4)?M. Nevertheless, the catalytic efficiency values (V (max)/K (m)) reached only a very small fraction of that with 3-aminopropanal (except for LeAMADH1 activity with two pyridine-derived compounds). Docking experiments using the crystal structure of PsAMADH2 were involved to discuss differences in results with position isomers or alkyl chain homologs.     

Identification of two residues essential for the stringent substrate specificity and active site stability of the prokaryotic l-arginine:glycine amidinotransferase CyrA

A novel prokaryotic l-arginine:glycine amidinotransferase (CyrA; EC2.1.4.1) is involved in the biosynthesis of the polyketide-derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J277, 3844-3860]. In order to elucidate the structure-function-stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human l-arginine:glycine amidinotransferase (h-AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h-AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand-induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand-induced structural changes that involve Phe245. Stability parameters (T(opt) and T(max) ) of the CyrA variants differed from those of wild-type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in T(opt) are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme.