Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the panE gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP(+) release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 +/- 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 +/- 0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by (1)H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation.     

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.     

Pantothenate production in Escherichia coli K12 by enhanced expression of the panE gene encoding ketopantoate reductase.

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. coli double mutant was employed to isolate the E. coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate.     

Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase

We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D (334) --> K and Y (335) --> F (factor V (KF)) and D (334) --> A and Y (335) --> A (factor V (AA)). Kinetic studies showed that while factor Va (KF) and factor Va (AA) had a K D for factor Xa similar to the K D observed for wild-type factor Va (factor Va (WT)), the clotting activities of the mutant molecules were impaired and the k cat of prothrombinase assembled with factor Va (KF) and factor Va (AA) was reduced. The second-order rate constant of prothrombinase assembled with factor Va (KF) or factor Va (AA) for prothrombin activation was approximately 10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor Va (WT). We also created quadruple mutants combining mutations in the amino acid region 334-335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E (323)Y (324) and E (330)V (331)). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor Va (WT). The data demonstrate that amino acid region 334-335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.     

Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.     

Double-mutant analysis of the interaction of Ras with the Ras-binding domain of RGL

RalGDS is a guanine nucleotide dissociation stimulator for Ral, and one of its homologues is RGL (RalGDS-like). In this study, the effects of mutations of Ras and the Ras-binding domains (RBDs) of RalGDS and RGL on their binding have been systematically examined. The D33A mutation of Ras reduces the abilities to bind RGL-RBD and RalGDS-RBD. To identify the RGL residue interacting with Asp33 of Ras, double-mutant analyses between Ras and RGL-RBD were conducted. For example, the K685A mutation of RGL-RBD has a much smaller effect on the RGL-RBD binding ability of the D33A mutant than on those of other mutants of Ras. Accordingly, it is indicated that the attractive interaction of Asp33 in Ras with Lys685 in RGL-RBD (Lys816 in RalGDS-RBD) contributes to the Ras.RBD association. This interaction is consistent with the crystal structure of the complex of RalGDS-RBD and the E31K Ras mutant [Huang, L., Hofer, F., Martin, G. S., and Kim, S.-H. (1998) Nat. Struct. Biol. 5, 422-426]. This crystal structure exhibits interactions of the mutation-derived Lys31 side chain with three RalGDS residues. Glu31 of Ras discriminates Ras from a Ras-homologue, Rap1, with Lys31, with respect to RalGDS and RGL binding; the E31K mutation of Ras potentiates the abilities to bind RGL-RBD and RalGDS-RBD. To examine the role of Glu31 of the wild-type Ras in the interaction with RGL and RalGDS, double-mutant analyses were conducted. The Ras binding ability of the E689A mutant of RGL-RBD is much stronger than that of the wild-type RGL-RBD, and the E31K mutation of Ras no longer potentiates the Ras binding ability of the E689A mutant. Therefore, the repulsive interaction between Glu31 in Ras and Glu689 in RGL-RBD (Asp820 in RalGDS-RBD) may keep the Ras.RBD association weaker than the Rap1.RBD association, which might be relevant to the regulation of the signaling network.     

Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity

We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.     

Reassessing the type I dehydroquinate dehydratase catalytic triad: Kinetic and structural studies of Glu86 mutants

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined—Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction—the role of Glu86 remains poorly characterized. To probe Glu86′s role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild‐type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.     

Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.     

Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of (D)V and (D(2)O)V for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, (2)H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys(159) changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on V(max) and V/K(androsterone) and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on V(max) is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Br?nsted relationship between the log(V/K(d)(-base)Et) of the external amine (corrected for molecular size effects) and pK(a) is linear for the K159A mutant-catalyzed reaction at pH 10.4 (beta = 0.85 +/- 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr(155) to Lys(159) via 2'-OH of ribose.     

The crystal structure of Escherichia coli ketopantoate reductase with NADP+ bound

The NADPH-dependent reduction of ketopantoate to pantoate, catalyzed by ketopantoate reductase (KPR; EC 1.1.1.169), is essential for the biosynthesis of pantothenate (vitamin B(5)). Here we present the crystal structure of Escherichia coli KPR with NADP(+) bound, solved to 2.1 A resolution. The cofactor is bound in the active site cleft between the N-terminal Rossmann-fold domain and the C-terminal alpha-helical domain. The thermodynamics of cofactor and substrate binding were characterized by isothermal titration calorimetry. The dissociation constant for NADP(+) was found to be 6.5 muM, 20-fold larger than that for NADPH (0.34 muM). The difference is primarily due to the entropic term, suggesting favorable hydrophobic interactions of the more lipophilic nicotinamide ring in NADPH. Comparison of this binary complex structure with the previously studied apoenzyme reveals no evidence for large domain movements on cofactor binding. This observation is further supported both by molecular dynamics and by calorimetric analysis. A model of the ternary complex, based on the structure presented here, provides novel insights into the molecular mechanism of enzyme catalysis. We propose a conformational switch of the essential Lys176 from the "resting" state observed in our structure to an "active" state, to bind ketopantoate. Additionally, we identify the importance of Asn98 for substrate binding and enzyme catalysis.     

Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis

The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap(4)A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap(4)A hydrolase fusion proteins were expressed and their k(cat) and K(m) values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type k(cat) of 23 s(-1) was reduced by 10(5)-, 10(3)-, and 30-fold, respectively, by replacement of the conserved P(4)-phosphate-binding catalytic residues Glu(56), Glu(52), and Glu(103) by Gln. K(m) values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His(31) to Val or Ala and Lys(83) to Met produced 10- and 16-fold increases in K(m) compared with the wild type value of 8.8 microm. These residues stabilize the P(1)-phosphate. H31V and H31A had a normal k(cat) but K83M showed a 37-fold reduction in k(cat). Lys(36) also stabilizes the P(1)-phosphate and a K36M mutant had a 10-fold reduced k(cat) but a relatively normal K(m). Thus both Lys(36) and Lys(83) may play a role in catalysis. The previously suggested roles of Tyr(27), His(38), Lys(79), and Lys(81) in stabilizing the P(2) and P(3)-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr(76) and Tyr(121), which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased K(m) 4-fold. It is concluded that interactions with the P(1)- and P(4)-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that it may have a much wider substrate range then previously believed.     

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Schizophyllum communealpha,alpha-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into alpha-d-glucose 1-phosphate and alpha-d-glucose. Recruitment of phosphate by the free enzyme induces alpha,alpha-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km=21,000 m(-1).s(-1)) was dramatic (>or=10(7)-fold) in purified Arg507-->Ala (R507A) and Lys512-->Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4-, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with alpha,alpha-trehalose (Ki=0.4 microm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki=300 microm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward alpha,alpha-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.