Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

Mutation of active site residues of the puromycin-sensitive aminopeptidase: conversion of the enzyme into a catalytically inactive binding protein

The active site glutamate, Glu 309, of the puromycin-sensitive aminopeptidase was mutated to glutamine, alanine, and valine. These mutants were characterized with amino acid beta-naphthylamides as substrates and dynorphin A(1-9) as an alternate substrate inhibitor. Conversion of glutamate 309 to glutamine resulted in a 5000- to 15,000-fold reduction in catalytic activity. Conversion of this residue to alanine caused a 25,000- to 100,000-fold decrease in activity, while the glutamate to valine mutation was the most dramatic, reducing catalytic activity 300,000- to 500,000-fold. In contrast to the dramatic effect on catalysis, all three mutations produced relatively small (1.5- to 4-fold) effects on substrate binding affinity. Mutation of a conserved tyrosine, Y394, to phenylalanine resulted in a 1000-fold decrease in k(cat), with little effect on binding. Direct binding of a physiological peptide, dynorphin A(1-9), to the E309V mutant was demonstrated by gel filtration chromatography. Taken together, these data provide a quantitative assessment of the effect of mutating the catalytic glutamate, show that mutation of this residue converts the enzyme into an inactive binding protein, and constitute evidence that this residue acts a general acid/base catalyst. The effect of mutating tyrosine 394 is consistent with involvement of this residue in transition state stabilization.     

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.     

Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.     

Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding

The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.     

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.     

Phenylalanine residues in the active site of tyrosine hydroxylase: mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300.

Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding. Also based on the structure, Phe300 has been reported to be hydroxylated due to a naturally occurring posttranslational modification [Goodwill, K. E., Sabatier, C., and Stevens, R. C. (1998) Biochemistry 37, 13437-13445]. Mutants of tyrosine hydroxylase with alanine substituted for Phe300 or Phe309 have now been purified and characterized. The F309A protein possesses 40% less activity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the production of dihydropterin. The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPA and dihydropterin. The K(6-MPH4) value for F300A tyrosine hydroxylase is twice the wild-type value. These results are consistent with Phe309 having a role in maintaining the integrity of the active site, while Phe300 contributes less than 1 kcal/mol to binding tetrahydropterin. Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolated catalytic domain after incubation with a large excess of 7, 8-dihydropterin, DTT, and Fe(2+). The modification is not observed in the untreated catalytic domain or in the full-length protein, even in the presence of excess iron. These results establish that hydroxylation of Phe300 is an artifact of the crystallography conditions and is not relevant to catalysis.     

The X-ray structure of N-methyltryptophan oxidase reveals the structural determinants of substrate specificity

The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage.     

Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.     

Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N(1)-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k(cat)/K(O2) value changes very little upon mutation with N(1)-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k(cat)/K(M)-pH profiles with N(1)-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK(a) values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK(a) as wild-type Fms1, about ～7.4; this pK(a) is assigned to the substrate N4. The k(cat)/K(O2)-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK(a) of about ～6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k(cat) value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 ?. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure.     

Mechanistic studies of mouse polyamine oxidase with N1,N12-bisethylspermine as a substrate

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k(cat) value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k(cat) value decreases below a pK(a) of 9.0. The k(cat)/K(m) value for BESPM exhibits a bell-shaped pH profile, with pK(a) values of 9.8 and 10.8. These pK(a) values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k(cat) value and the k(cat)/K(m) value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.     

The role of water in the catalytic efficiency of triosephosphate isomerase

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.     

An aspartate residue at the extracellular boundary of TMII and an arginine residue in TMVII of the gastrin-releasing peptide receptor interact to facilitate heterotrimeric G protein coupling.

The mammalian bombesin receptor subfamily of G protein-coupled receptors currently consists of the gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor, and bombesin receptor subtype 3. All three receptors contain a conserved aspartate residue (D98) at the extracellular boundary of transmembrane domain II and a conserved arginine residue (R309) near the extracellular boundary of transmembrane domain VII. To evaluate the functional role of these residues, site-directed GRP-R mutants were expressed in fibroblasts and assayed for their ability to both bind agonist and catalyze exchange of guanine nucleotides. Alanine substitution at GRP-R position 98 or 309 reduced agonist binding affinity by 24- and 56-fold, respectively, compared to wild-type GRP-R. Single swap GRP-R mutations either resulted in no receptor expression in the membrane (D98R) or the protein was not able to bind agonist (R309D). In contrast, the double swap mutation (D98R/R309D) had high-affinity agonist binding, reduced from wild-type GRP-R by only 6-fold. In situ reconstitution of urea-extracted membranes expressing either wild-type or mutant (D98A or R309A) GRP-R with G(q) indicated that alanine substitution greatly reduced G protein catalytic exchange compared to wild-type GRP-R. The D98R/R309D GRP-R had both a higher intrinsic basal activity and a higher overall catalytic exchange activity compared to wild-type; however, the wild-type GRP-R produced a larger agonist-stimulated response relative to the double swap mutant. Taken together, these data show that GRP-R residues D98 and R309 are critical for efficient coupling of GRP-R to G(q). Furthermore, our findings are consistent with a salt bridge interaction between these two polar and oppositely charged amino acids that maintains the proper receptor conformation necessary to interact with G proteins.     

A catalytic mechanism that explains a low catalytic activity of serine dehydratase like-1 from human cancer cells: crystal structure and site-directed mutagenesis studies

SDH (l-serine dehydratase, EC 4.3.1.17) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes dehydration of l-Ser/Thr to yield pyruvate/ketobutyrate and ammonia. A SDH isoform (cSDH) found in human cancer cell lines has relatively low catalytic activity in comparison with the liver enzyme (hSDH). The crystal structure of cSDH has been determined at 2.8 angstroms resolution. A PLP is covalently attached to K48 by Schiff-base linkage in the active site. The ring nitrogen of PLP is involved in a H-bonding with C309, but is apparently not protonated. Twenty-three amino residues that compose the active site surfaces were identified. The human and rat liver enzymes (hSDH and rSDH) have the same residues, while residues G72, A172, and S228 in cSDH are replaced with A66, S166, and A222, respectively, in hSDH. These residues in hSDH and cSDH were mutated to make complementary pairs of mutated enzymes, and their kinetic parameters were determined. C303 of hSDH and C309 of cSDH which are H-bonding partner of the ring nitrogen of PLP were mutated to alanine and their kinetic parameters were also determined. The crystal structures and the mutation data suggest that having a glycine at residue 72 of cSDH is the major reason for the reduction of catalytic activity of cSDH. Changing alanine to glycine at residue 72 increases the flexibility of the substrate binding-loop (71S(G/A)GN74), so that the bound substrate and PLP are not pushed deep into the active cleft. Consequently, the proton transfer rate from S(G) of C309 to N1 of the bound PLP is decreased, which determines the rate of catalytic reaction.     

Effect of an E461G mutation of beta-galactosidase (Escherichia coli, lac Z) on pL rate profiles and solvent deuterium isotope effects.

An E461G mutation of beta-galactosidase results in the disappearance of the high pL (L = H, D) downward break in the rate profiles for k(cat)/K(m) for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl beta-D-galactopyranoside (Gal-OPNP) and a decrease from (k(cat))(HOH)/(k(cat))(DOD) = 1.7 to (k(cat))(HOH)/(k(cat))(DOD) = 1.2 in the solvent deuterium isotope effect. These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free beta-galactosidase and they are consistent with a role for this residue in Br?nsted acid catalysis at the leaving group. The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for k(s) for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the beta-D-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the beta-D-galactopyranosyl reaction intermediate.     

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.     

Differential contributions of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis

Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg(454), Tyr(456), Cys(472), Gly(488), Thr(491), and Trp(677)) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr(456), which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4'-hydroxyl, or Arg(454), which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity. In contrast, substitution of Thr(491), which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456S K(m)(cyt )(c) and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp(677) is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp(677) and the carboxyl-terminal Ser(678) decreased catalytic activity 50-fold without affecting FAD content.     

Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A

The FAD binding site of human liver monoamine oxidase A (MAO A) has been investigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme is labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm. The results from both in vivo and in vitro reconstitution experiments show increased activity levels (up to approximately 7-fold higher) with those analogues exhibiting higher oxidation-reduction potentials than normal flavin and decreased activity levels with analogues exhibiting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific interactions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional significance of 8alpha-covalent binding of flavins to proteins.     

Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate.

The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.