Isocyanide binding to the copper(I) centers of the catalytic core of peptidylglycine monooxygenase (PHMcc)

Binding of the Cu(I)-specific ligands 2,6-dimethylphenyl isocyanide (DIMPI) and isopropyl isocyanide (IPI) to the reduced form of peptidylglycine monooxygenase (PHM) is reported. Both ligands bind to the methionine-containing CuM center, eliciting FTIR bands at 2,138 and 2,174 cm(-1), respectively, but appear unable to coordinate at the histidine-containing CuH center in the wild-type enzyme. This chemistry parallels that previously observed for CO binding to the reduced PHM catalytic core (PHMcc). However, in contrast to the CO chemistry, peptide substrate binding did not induce binding of the isocyanide at CuH. XAS confirmed the binding of DIMPI at CuM via the observation of a short Cu-C interaction at 1.87 A and by the lengthening of the Cu-S(methionine) bond length by 0.06 A. Similarly, FTIR studies on DIMPI binding to the M314I and H172A mutant forms of reduced PHMcc confirmed the assignment of the 2,138-cm(-1) IR band as a CuM-DIMPI complex, but surprisingly also showed DIMPI binding to CuH, as indicated by a band at 2,148 cm(-1). An inorganic complex, [Cu(1,2-Me2Im)2(DIMPI)](PF6), was synthesized and its crystal structure was determined as a model for the interaction of isocyanides with imidazole-containing Cu(I) complexes. Comparison of EXAFS data for the protein and model suggests that DIMPI probably binds to CuM in a tilted fashion, similar to that of ethyl isocyanide binding to myoglobin.     

Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper

A derivative of peptidylglycine monooxygenase which lacks the CuH center has been prepared and characterized. This form of the enzyme is termed the half-apo protein. Copper-to-protein stoichiometric measurements establish that the protein binds only one of the two copper centers (CuM and CuH) found in the native enzyme. Confirmation that the methionine-containing CuM has been retained has been obtained from EXAFS experiments which show that the characteristic signature of the Cu-S(Met) interaction is preserved. The half-apo derivative binds 1 equiv of CO per copper with an IR frequency of 2092 cm(-1), and this monocarbonyl also displays the Cu-S(Met) interaction in its EXAFS spectrum. These results allow unambiguous assignment of the 2092 cm(-1) band as a CuM-CO species. Binding of CO in the presence of peptide substrate was also investigated. In the native enzyme, substrate induced binding of a second CO molecule with an IR frequency of 2062 cm(-1), tentatively assigned to a CO complex of the histidine-containing CuH site. Unexpectedly, this reactivity is also observed in the half-apo derivative, although the intensity distribution of the CO stretches now indicates that the copper has been partially transferred to a second site, believed to be CuH. The implications of this observation are discussed in terms of a possible additional peptide binding site close to the CuH center.     

A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK(a) of 4.6 to generate the inactive low-pH form with Cu(H) coordinated by M109, H107, and H172.     

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.     

Role of His505 in the soluble fumarate reductase from Shewanella frigidimarina

The X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] clearly shows the presence of an internally bound sodium ion. This sodium ion is coordinated by one solvent water molecule (Wat912) and five backbone carbonyl oxygens from Thr506, Met507, Gly508, Glu534, and Thr536 in what is best described as octahedral geometry (despite the rather long distance from the sodium ion to the backbone oxygen of Met507 (3.1 A)). The water ligand (Wat912) is, in turn, hydrogen bonded to the imidazole ring of His505. This histidine residue is adjacent to His504, a key active-site residue thought to be responsible for the observed pK(a) of the enzyme. Thus, it is possible that His505 may be important in both maintaining the sodium site and in influencing the active site. Here we describe the crystallographic and kinetic characterization of the H505A and H505Y mutant forms of the Shewanella fumarate reductase. The crystal structures of both mutant forms of the enzyme have been solved to 1.8 and 2.0 A resolution, respectively. Both show the presence of the sodium ion in the equivalent position to that found in the wild-type enzyme. The structure of the H505A mutant shows the presence of two water molecules in place of the His505 side-chain which form part of a hydrogen-bonding network with Wat48, a ligand to the sodium ion. The structure of the H505Y mutant shows the hydroxyl group of the tyrosine side-chain hydrogen-bonding to a water molecule which is also a ligand to the sodium ion. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the mutant enzymes are catalytically active but show markedly different pH profiles compared to the wild-type enzyme. At high pH (above 8.5), the wild type and mutant enzymes have very similar activities. However, at low pH (6.0), the H505A mutant enzyme is some 20-fold less active than wild-type. The combined crystallographic and kinetic results suggest that His505 is not essential for sodium binding but does affect catalytic activity perhaps by influencing the pK(a) of the adjacent His504.     

Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease

Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.     

Switching from an Induced-Fit to a Lock-and-Key Mechanism in an Aminoacyl-tRNA Synthetase with Modified Specificity

Methionyl-tRNA synthetase (MetRS) specifically binds its methionine substrate in an induced-fit mechanism, with methionine binding causing large rearrangements. Mutated MetRS able to efficiently aminoacylate the methionine (Met) analog azidonorleucine (Anl) have been identified by saturation mutagenesis combined with in vivo screening procedures. Here, the crystal structure of such a mutated MetRS was determined in the apo form as well as complexed with Met or Anl (1.4 to 1.7 Å resolution) to reveal the structural basis for the altered specificity. The mutations result in both the loss of important contacts with Met and the creation of new contacts with Anl, thereby explaining the specificity shift. Surprisingly, the conformation induced by Met binding in wild-type MetRS already occurs in the apo form of the mutant enzyme. Therefore, the mutations cause the enzyme to switch from an induced-fit mechanism to a lock-and-key one, thereby enhancing its catalytic efficiency.     

The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase (PHM): a spectroscopic study of the H172A mutant

The spectroscopic characterization of the H172A mutant of peptidylglycine alpha-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu(H) ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 A in wild-type PHM to 1.89 A in H172A associated with a change in the structure of Cu(H) from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu(M) site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu(H) was obtained through the observation that loss of the H172 Cu(H) ligand caused a 3 cm(-)(1) blue shift in the nu(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.     

Modulation of the remote heme site geometry of recombinant mouse neuronal nitric-oxide synthase by the N-terminal hook region

The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated. Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems. The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wild-type enzyme. Replacement of Asp-314 by Ala altered the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogated the ability of nNOS to form catalytically active dimers. Replacement of Thr-315 by Ala reduced the protein stability and altered the geometry around the heme site, especially in the absence of bound (6R)-5,6,7, 8-tetrahydro-L-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS.     

The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.     

Structural analysis of flavinylation in vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.     

Role of Lys-226 in the catalytic mechanism of Bacillus stearothermophilus serine hydroxymethyltransferase--crystal structure and kinetic studies

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the reversible conversion of l-Ser and tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural studies on this enzyme have implicated several residues in the catalytic mechanism, one of them being the active site lysine, which anchors PLP. It has been proposed that this residue is crucial for product expulsion. However, in other PLP-dependent enzymes, the corresponding residue has been implicated in the proton abstraction step of catalysis. In the present investigation, Lys-226 of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to evaluate the role of this residue in catalysis. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff base formation with lysine is not essential for PLP binding. The 3D structure of the mutant enzymes revealed that PLP was bound at the active site in an orientation different from that of the wild-type enzyme. In the presence of substrate, the PLP ring was in an orientation superimposable with that of the external aldimine complex of wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic analysis of the different steps of catalysis revealed that there was a drastic reduction in the rate of formation of the quinonoid intermediate. Analysis of these results along with the crystal structures suggested that K-226 is responsible for flipping of PLP from one orientation to another which is crucial for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.     

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.     

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.     

Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation

The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.     

Methionine-141 directly influences the binding of 4-methylpyrazole in human sigma sigma alcohol dehydrogenase

Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human sigma sigma isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human beta1beta1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4-methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human sigma sigma alcohol dehydrogenase (ADH) isoenzyme to 2.5 A resolution. Comparison of this structure to the human beta1beta1 isoenzyme structure indicated that the side-chain position for Met141 in sigma sigma ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in sigma sigma ADH to Leu (sigma141L) lowers the Ki for 4-methylpyrazole from 350 to 10 microM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate-binding pocket at a position close to the methyl group of 4-methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X-ray structure of the sigma141L mutant enzyme was determined to 2.4 A resolution. The three-dimensional structure of the mutant enzyme is identical to the wild-type enzyme, with the exception of the residue at position 141. Thus, the differences in 4-methylpyrazole binding between the mutant and wild-type sigma sigma ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class-specific differences in 4-methylpyrazole binding to the human ADH isoenzymes.     

Implication by site-directed mutagenesis of Arg314 and Tyr316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase

Sequence alignment of pig mitochondrial NADP-dependent isocitrate dehydrogenase with eukaryotic (human, rat, and yeast) and Escherichia coli isocitrate dehydrogenases reveals that Tyr316 is completely conserved and is equivalent to the E. coli Tyr345, which interacts with the 2'-phosphate of NADP in the crystal structure [Hurley et al., Biochemistry 30 (1991) 8671-8678]. Lys321 is also completely conserved in the five isocitrate dehydrogenases. Either an arginine or lysine residue is found among the enzymes from other species at the position corresponding to the pig enzyme Arg314. While Arg323 is not conserved among all species, its proximity to the coenzyme site makes it a good candidate for investigation. The importance of these four amino acids to the function of pig mitochondrial NADP-isocitrate dehydrogenase was studied by site-directed mutagenesis. Mutants (R314Q, Y316F, Y316L, K321Q, and R323Q) were generated by a megaprimer polymerase chain reaction method. Wild-type and mutant enzymes were expressed in E. coli and purified to homogeneity. All mutant and wild-type enzymes exhibited comparable molecular weights indicative of the dimeric enzyme. Mutations do not cause an appreciable change in enzyme secondary structure as revealed by circular dichroism measurements. The kinetic parameters (V(max) and K(M) values) of K321Q and R323Q are similar to those of wild-type, indicating that Lys321 and Arg323 are not involved in enzyme function. R314Q exhibits a 10-fold increase in K(M) for NADP as compared to that of wild-type, while they have comparable V(max) values. These results suggest that Arg314 contributes to the affinity between the enzyme and NADP. The hydroxyl group of Tyr316 is not required for enzyme function since Y316F exhibits similar kinetic parameters to those of wild-type. Y316L shows a 4-fold increase in K(M) for NADP and a decrease in V(max) as compared to wild-type, suggesting that the aromatic ring of the Tyr of isocitrate dehydrogenase contributes to the affinity for coenzyme, as well as to catalysis. The K(i) for NAD of R314Q, Y316F, and Y316L is comparable to that of wild-type, indicating that the Arg314 and Tyr316 may be located near the 2'-phosphate of enzyme-bound NADP.     

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0–2.8 Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (βα)₈-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.     

Replacement of Asp-162 by Ala prevents the cooperative transition by the substrates while enhancing the effect of the allosteric activator ATP on E. coli aspartate transcarbamoylase

The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states. To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine. The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme. Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme. Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern. In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure. Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution. This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model. Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.