Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.     

Homology modeling of the insect chitinase catalytic domain--oligosaccharide complex and the role of a putative active site tryptophan in catalysis

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell-line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.     

Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA

The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k(cat), while more noticeable affects were observed in K(m). The mutant D370A/Y540F showed the most significant changes in the pH dependence of k(cat)/K(m) and K(m) relative to wild-type PutA86-601 with an apparent pK(a) value of about 8.2 for the pH-dependent decrease in K(m). From the pH profile of D370A/Y540F inhibition by l-tetrahydro-2-furoic acid (l-THFA), the pH dependency of K(m) in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the E(m) for bound FAD. At pH 7.0, E(m) values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in E(m) determined for D370A relative to wild-type PutA86-601 (E(m)=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.     

Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli

The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid. We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J. (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine. After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots. The mature enzyme and the Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney. The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide. Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding. These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.     

Pleiotropic impact of a single lysine mutation on biosynthesis of and catalysis by N-methyltryptophan oxidase

N-Methyltryptophan oxidase (MTOX) contains covalently bound FAD. N-Methyltryptophan binds in a cavity above the re face of the flavin ring. Lys259 is located above the opposite, si face. Replacement of Lys259 with Gln, Ala, or Met blocks (>95%) covalent flavin incorporation in vivo. The mutant apoproteins can be reconstituted with FAD. Apparent turnover rates (k(cat,app)) of the reconstituted enzymes are ~2500-fold slower than those of wild-type MTOX. Wild-type MTOX forms a charge-transfer E(ox)·S complex with the redox-active anionic form of NMT. The E(ox)·S complex formed with Lys259Gln does not exhibit a charge-transfer band and is converted to a reduced enzyme·imine complex (EH(2)·P) at a rate 60-fold slower than that of wild-type MTOX. The mutant EH(2)·P complex contains the imine zwitterion and exhibits a charge-transfer band, a feature not observed with the wild-type EH(2)·P complex. Reaction of reduced Lys259Gln with oxygen is 2500-fold slower than that of reduced wild-type MTOX. The latter reaction is unaffected by the presence of bound product. Dissociation of the wild-type EH(2)·P complex is 80-fold slower than k(cat). The mutant EH(2)·P complex dissociates 15-fold faster than k(cat,app). Consequently, EH(2)·P and free EH(2) are the species that react with oxygen during turnover of the wild-type and mutant enzyme, respectively. The results show that (i) Lys259 is the site of oxygen activation in MTOX and also plays a role in holoenzyme biosynthesis and N-methyltryptophan oxidation and (ii) MTOX contains separate active sites for N-methyltryptophan oxidation and oxygen reduction on opposite faces of the flavin ring.     

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of p-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site-to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of p-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis.     

Hyperactive mutants of mouse d-aspartate oxidase: mutagenesis of the active site residue serine 308

The role of Ser-308 of murine D-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards D-Asp and N-methyl-D-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.     

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.     

Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268

Carp muscle-specific creatine kinase M1 isoenzyme (M1-CK) seems to have evolved to adapt to synchronized changes in body temperature and intracellular pH. When gly(268) in rabbit muscle-specific creatine kinase was substituted with asn(268) as found in carp M1-CK, the rabbit muscle-specific CK G286N mutant specific activity at pH 8.0 and 10°C was more than 2-fold higher than that in the wild-type rabbit enzyme. Kinetic studies showed that K(m) values of the rabbit CK G268N mutant were similar to those of the wild-type rabbit enzyme, yet circular dichroism spectra showed that the overall secondary structures of the mutant enzyme, at pH 8.0 and 5°C, were almost identical to the carp M1-CK enzyme. The X-ray diffraction pattern of the mutant enzyme crystal revealed that amino acid residues involved in substrate binding are closer to one another than in the rabbit enzyme, and the cysteine283 active site of the mutant enzyme points away from the ADP binding site. At pH 7.4-8.0 and 35-10°C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the wild-type rabbit enzyme and more similar to the carp M1-CK enzyme. Thus, the smaller active site of the RM-CK G268N mutant may be one of the reasons for its improved activity at low temperature.     

Assimilatory nitrate reductase: lysine 741 participates in pyridine nucleotide binding via charge complementarity

The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the activity of a P450 2E1 mutant where the conserved threonine at position 303 was replaced with an alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K(I) of 1.6 microM. PEITC also inactivated the wild-type P450 2E1 as efficiently with a K(I) of 2.7 microM. The inactivation was entirely dependent on NADPH and followed pseudo-first-order kinetics. Previously we reported the mechanism-based inactivation of wild-type P450 2E1 by BITC with a K(I) of 13 microM. In contrast to the wild-type enzyme, the P450 2E1 T303A mutant was not inactivated by BITC but it was inhibited in a competitive manner with a K(i) of 3 microM. The binding constants determined by spectral binding studies were similar for both enzymes. The binding of BITC produced characteristic Type I spectral changes in the wild-type and mutant enzyme. A radiolabeled BITC metabolite bound to P450 2E1 and to P450 2E1 T303A when both enzymes were incubated with [(14)C]BITC and NADPH. Whole protein electrospray ion trap mass spectrometry indicated that a mass consistent with one molecule of benzylisocyanate and oxygen was adducted to the wild-type enzyme. The mass adducted to the T303A mutant was consistent with the addition of one hydroxylated BITC or of one benzylisocyanate moiety and one sulfur molecule. Analysis of the metabolites of BITC indicated that each enzyme produced similar metabolites but that the mutant enzyme generated significantly higher amounts of benzaldehyde and benzoic acid when compared to the wild-type enzyme.     

Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase

Chitinase is an enzyme used by insects to degrade the structural polysaccharide, chitin, during the molting process. Tryptophan 145 (W145) of Manduca sexta (tobacco hornworm) chitinase is a highly conserved residue found within a second conserved region of family 18 chitinases. It is located between aspartate 144 (D144) and glutamate 146 (E146), which are putative catalytic residues. The role of the active site residue, W145, in M. sexta chitinase catalysis was investigated by site-directed mutagenesis. W145 was mutated to phenylalanine (F), tyrosine (Y), isoleucine (I), histidine (H), and glycine (G). Wild-type and mutant forms of M. sexta chitinases were expressed in a baculovirus-insect cell line system. The chitinases secreted into the medium were purified and characterized by analyzing their catalytic activity and substrate or inhibitor binding properties. The wild-type chitinase was most active in the alkaline pH range. Several of the mutations resulted in a narrowing of the range of pH over which the enzyme hydrolyzed the polymeric substrate, CM-Chitin-RBV, predominantly on the alkaline side of the pH optimum curve. The range was reduced by about 1 pH unit for W145I and W145Y and by about 2 units for W145H and W145F. The W145G mutation was inactive. Therefore, the hydrophobicity of W145 appears to be critical for maintaining an abnormal pKa of a catalytic residue, which extends the activity further into the alkaline range. All of the mutant enzymes bound to chitin, suggesting that W145 was not essential for binding to chitin. However, the small difference in Km's of mutated enzymes compared to Km values of the wild-type chitinase towards both the oligomeric and polymeric substrates suggested that W145 is not essential for substrate binding but probably influences the ionization of a catalytically important group(s). The variations in kcat's among the mutated enzymes and the IC50 for the transition state inhibitor analog, allosamidin, indicate that W145 also influences formation of the transition state during catalysis.     

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for G_M1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, G_Sα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.     

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.     

Properties of p-hydroxybenzoate hydroxylase when stabilized in its open conformation

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently, it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea, we have studied the properties of two different mutant forms of the enzyme that are stabilized in the open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis.     

Electron transfer in flavocytochrome P450 BM3: kinetics of flavin reduction and oxidation,the role of cysteine 999, and relationships with mammalian cytochrome P450 reductase

Cys-999 is one component of a triad (Cys-999, Ser-830, and Asp-1044) located in the FAD domain of flavocytochrome P450 BM3 that is almost entirely conserved throughout the diflavin reductase family of enzymes. The role of Cys-999 has been studied by steady-state kinetics, stopped-flow spectroscopy, and potentiometry. The C999A mutants of BM3 reductase (containing both FAD and FMN cofactors) and the isolated FAD domain are substantially compromised in their capacity to reduce artificial electron acceptors in steady-state turnover with either NADPH or NADH as electron donors. Stopped-flow studies indicate that this is due primarily to a substantially slower rate of hydride transfer from nicotinamide coenzyme to FAD cofactor in the C999A enzymes. The compromised rates of hydride transfer are not attributable to altered thermodynamic properties of the flavins. A reduced enzyme-NADP(+) charge-transfer species is populated following hydride transfer in the wild-type FAD domain, consistent with the slow release of NADP(+) from the 2-electron-reduced enzyme. This intermediate does not accumulate in the C999A FAD domain or wild-type and C999A BM3 reductases, suggesting more rapid release of NADP(+) from these enzyme forms. Rapid internal electron transfer from FAD to FMN in wild-type BM3 reductase releases NADP(+) from the nicotinamide-binding site, thus preventing the inhibition of enzyme activity through the accumulation of a stable FADH(2)-NADP(+) charge-transfer complex. Hydride transfer is reversible, and the observed rate of oxidation of the 2-electron-reduced C999A BM3 reductase and FAD domain is hyperbolically dependent on NADP(+) concentration. With the wild-type BM3 reductase and FAD domain, the rate of flavin oxidation displays an unusual dependence on NADP(+) concentration, consistent with a two-site binding model in which two coenzyme molecules bind to catalytic and regulatory regions (or sites) within a bipartite coenzyme binding site. A kinetic model is proposed in which binding of coenzyme to the regulatory site hinders sterically the release of NADPH from the catalytic site. The results are discussed in the light of kinetic and structural studies on mammalian cytochrome P450 reductase.     

Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity.

NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors. Residues Tyr140 and Tyr178 are known to be involved in FMN binding. A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role. This role has been investigated by site-directed mutagenesis. Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50%. Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed. The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined. The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants. Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex. The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site. The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.     

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability

NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.