Flavin conformational changes in the catalytic cycle of p-hydroxybenzoate hydroxylase substituted with 6-azido- and 6-aminoflavin adenine dinucleotide

Crystallographic studies have demonstrated two flavin conformations for p-hydroxybenzoate hydroxylase (PHBH) [Gatti, D. L., Palfey, B. A. , Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., & Ludwig, M. L. (1994) Science 266, 110-114. Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., & van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. The isoalloxazine ring system of one conformation (the "out" conformation) is significantly more exposed to solvent and is not in position for necessary catalytic reactions, but when the natural substrate is bound to the enzyme, the isoalloxazine is in the correct position (the "in" conformation) for its chemical function. In this study, several aspects of the function of the conformational change in catalysis were explored using the wild-type and Tyr222Phe forms of PHBH substituted with 6-azido FAD. This flavin served as both a spectral probe and a photolabel. The enzyme containing 6-azido FAD was a relatively effective catalyst for the hydroxylation of p-hydroxybenzoate. However, the intermediate reduced 6-azido enzyme was chemically unstable, and a small fraction converted to 6-amino PHBH by the elimination of N2 during each catalytic cycle. The reduction of 6-azido FAD PHBH by NADPH was almost as fast as the reduction of the natural enzyme. The characteristic spectral change caused by NADPH binding prior to hydride transfer strongly suggests that flavin movement from the "in" to the "out" conformation precedes flavin reduction. Irradiation of 6-azido PHBH with visible light covalently labeled proline 293, an active site residue, under conditions in which the flavin adopted the "in" conformation, while no protein labeling occurred under conditions in which the flavin was "out". The labeled protein exchanged substrate and was reduced by NADPH much more slowly than before photolysis. It is therefore concluded that isoalloxazine movement is required for pyridine nucleotide to gain access to the active site and for the exchange of aromatic ligands.     

Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding

The conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3-OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence-comparison studies suggest that Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH.     

Substrate recognition by "password" in p-hydroxybenzoate hydroxylase

The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [Gatti, D. L., Palfey, B. A., Lah, M.-S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science 266, 110-114; Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. Kinetic studies detected the movement of the flavin from the buried conformation to the exposed conformation caused by the binding of NADPH prior to its reaction with the flavin. The pH dependence of the rate constant for flavin reduction in wild-type PHBH and the His72Asn mutant indicates that the deprotonation of bound p-hydroxybenzoate is also required for flavin movement, and is accomplished by the same internal proton transport network previously found to be involved in substrate oxidation. The linkage of substrate deprotonation to flavin movement constitutes a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.     

The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis

BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols. RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction. CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.     

The catalytic domain of human immunodeficiency virus integrase: ordered active site in the F185H mutant

We solved the structure and traced the complete active site of the catalytic domain of the human immunodeficiency virus type 1 integrase (HIV-1 IN) with the F185H mutation. The only previously available crystal structure, the F185K mutant of this domain, lacks one of the catalytically important residues, E152, located in a stretch of 12 disordered residues [Dyda et al. (1994) Science 266, 1981-1986]. It is clear, however, that the active site of HIV-1 IN observed in either structure cannot correspond to that of the functional enzyme, since the cluster of three conserved carboxylic acids does not create a proper metal-binding site. The conformation of the loop was compared with two different conformations found in the catalytic domain of the related avian sarcoma virus integrase [Bujacz et al. (1995) J. Mol. Biol. 253, 333-346]. Flexibility of the active site region of integrases may be required in order for the enzyme to assume a functional conformation in the presence of substrate and/or cofactors.     

Distribution of nociceptive neurons in the ventrobasal complex of macaque thalamus

Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.     

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-A resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.     

The polymer basis of kinetics and equilibria of enzymes: the accessible-volume origin of entropy changes in a class Abeta-lactamase

The occurrence of enzymatic catalysis, as for any chemical reaction, depends critically upon close contact of the reactants, since making/breaking of bonds occurs over distances of about 0.2 A. Unlike small molecules, each enzyme molecule acts as an ordered solvent and reactant. Each group important to the enzyme reaction interacts with the substrate, then moves away, and subsequently binds another substrate. In other words, the group undergoes round trips in structure. For a round trip, the thermochemical state functions deltaG, deltaH, deltaS, etc., are zero. As a consequence, control of the binding of substrate must reside in the nonbinding conformations of the polymer since they govern the different fractions of time the macromolecule is in the correct conformation for bonding. Applying standard macromolecular models to the enzymes suggests that the majority of free energy for an enzyme reaction resides in the enzyme structure as an entropic contribution. Enthalpic contributions come from bond formation with the substrates and substrate structural changes. Further, it is shown that the molecular mechanisms that can effect binding and allosteric control fall into only three classes. Three x-ray structures of class A beta-lactamases (native, mutant, and with substrate) show the individual binding groups at the active site change their accessible volumes depending on substrate binding and mutant form. From these volume differences, the deltaS of reaction is calculated. The x-ray-derived deltaG = - TdeltaS matches the deltaG = -RT ln k1 from changes in rate constants for the same set of beta-penicillinases.     

A core folding model for catalysis by the hammerhead ribozyme accounts for its extraordinary sensitivity to abasic mutations

Introducing abasic nucleotides at each of 13 positions in the conserved core of the hammerhead ribozyme causes a large decrease in the extent of catalysis [Peracchi, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522]. This extreme sensitivity to structural defects is in contrast to the behavior of protein enzymes and larger ribozymes. Several additional differences in the behavior of the hammerhead relative to that of protein enzymes and larger ribozymes are described herein. The deleterious effects of the abasic mutations are not relieved by lowering the temperature, by increasing the concentration of monovalent or divalent metal ions, or by adding polyamines, in contrast to effects observed with protein enzymes and large RNA enzymes. In addition, the abasic mutations do not significantly weaken substrate binding. These results and previous observations are all accounted for by a "core folding" model in which the stable ground state structure of the hammerhead ribozyme complexed with the substrate is a partially folded state that must undergo an additional folding event to achieve its catalytic conformation. We propose that the peculiar behavior of the hammerhead arises because the limited structural interconnections in a small RNA enzyme do not allow the ground state to stably adopt the catalytic conformation; within the globally folded catalytic conformation, limited structural interconnections may further impair catalysis by hampering the precise alignment of active site functional groups. This behavior represents a basic manifestation of the well-recognized interconnection between folding and catalysis.     

Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the C1 beta of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher Kcat/Km for 1-chlorohexane and a 2-fold higher Kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in Kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.     

Active site of dihydroorotate dehydrogenase A from Lactococcus lactis investigated by chemical modification and mutagenesis

The flavin-containing enzyme dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate (DHO) to orotate, the first aromatic intermediate in pyrimidine biosynthesis. The first structure of a DHOD, the A form of the enzyme from Lactococcus lactis, has recently become known, and some conserved residues were suggested to have a role in the active site [Rowland et al. (1997) Structure 2, 239-252]. In particular, Cys 130 was hypothesized to work as a base, which activates dihydroorotate (DHO) for hydride transfer. By chemical modification and site-directed mutagenesis we have obtained results consistent with this proposal. Cys 130 was susceptible to alkylating reagents, and mutants of Cys 130 (C130A and C130S) showed hardly detectable enzyme activity at pH 8.0, while at pH 10 the C130S mutant enzyme had approximately 1% of wild-type activity. Mutants of Lys 43, Asn 132, and Lys 164 were also constructed. Exchange of Lys 43 to Ala or Glu (K43A and K43E) and of Asn 132 to Ala (N132A) affected both catalysis and substrate binding. Expressed as kcat/KM for DHO, the deterioration of these three mutant enzymes was 10(3)-10(4)-fold. Flavin spectra of the mutant enzymes were not, like the wild-type enzyme, bleached by DHO in stopped-flow experiments, showing that they were deficient with respect to the first half-reaction, namely reduction of FMN by DHO, which was not rate limiting for the wild-type enzyme. The binding interaction between flavin and the reaction product, orotate, could be monitored by a red shift of the flavin absorbance in the wild-type enzyme. The C130A, C130S, and N132A mutant enzymes displayed similar capacity to bind orotate. In contrast, orotate did not change the absorption spectra of the K43 mutant enzymes, although it did inhibit their activity. All of the mutant enzymes, except K164A, contained normal levels of flavin. The results are discussed in relation to the structures of DHODA and other flavoenzymes. The possible acid-base chemistry of Cys 130 is compared to previous work on mammalian dihydropyrimidine dehydrogenases, flavoenzymes, which catalyze the reversed reaction, namely the reduction of pyrimidine bases.     

Active site modifications in a double mutant of liver alcohol dehydrogenase: structural studies of two enzyme-ligand complexes

The oxidation of alcohol to aldehyde by horse liver alcohol dehydrogenase (LADH) requires the transfer of a hydride ion from the alcohol substrate to the cofactor nicotinamide adenine dinucleotide (NAD). A quantum mechanical tunneling contribution to this hydride transfer step has been demonstrated in a number of LADH mutants designed to enhance or diminish this effect [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. The active site double mutant Phe93 --> Trp/Val203 --> Ala shows a 75-fold reduction in catalytic efficiency relative to that of the native enzyme, and reduced tunneling relative to that of either single mutant. We present here two crystal structures of the double mutant: a 2.0 A complex with NAD and the substrate analogue trifluoroethanol and a 2.6 A complex with the isosteric NAD analogue CPAD and ethanol. Changes at the active site observed in both complexes are consistent with reduced activity and tunneling. The NAD-trifluoroethanol complex crystallizes in the closed conformation characteristic of the active enzyme. However, the NAD nicotinamide ring rotates away from the substrate, toward the space vacated by replacement of Val203 with the smaller alanine. Replacement of Phe93 with the larger tryptophan also produces unfavorable steric contacts with the nicotinamide carboxamide group, potentially destabilizing hydrogen bonds required to maintain the closed conformation. These contacts are relieved in the second complex by rotation of the CPAD pyridine ring into an unusual syn orientation. The resulting loss of the carboxamide hydrogen bonds produces an open conformation characteristic of the apoenzyme.     

Reaction of the C30A mutant of trimethylamine dehydrogenase with diethylmethylamine

The role played by the 6-S-cysteinyl-FMN bond of trimethylamine dehydrogenase in the reductive half-reaction of the enzyme has been studied by following the reaction of the slow substrate diethylmethylamine with a C30A mutant of the enzyme lacking the covalent flavin attachment to the polypeptide. Removal of the 6-S-cysteinyl-FMN bond diminishes the limiting rate for the first of the three observed kinetic phases of the reaction by a factor of 6, but has no effect on the rate constants for the two subsequent kinetic phases. The flavin in the C30A enzyme recovered from the reaction of the C30A enzyme with excess substrate is found to have been converted to the 6-hydroxy derivative, rendering the enzyme inactive. The noncovalently bound FMN of the C30A mutant enzyme is also converted to 6-hydroxy-FMN and rendered inactive upon reduction with excess trimethylamine, but not by reduction with dithionite, even at high pH or in the presence of the effector tetramethylammonium chloride. These results suggest that one significant role of the 6-S-cysteinyl-FMN bond is to prevent the inactivation of the enzyme during catalysis. A reaction mechanism is proposed whereby OH- attacks C-6 of a flavin-substrate covalent adduct in the course of steady-state turnover to form 6-hydroxy-FMN.     

Synthesis and use of iodinated nonsteroidal antiinflammatory drug analogs as crystallographic probes of the prostaglandin H2 synthase cyclooxygenase active site

The cyclooxygenase activity of the membrane protein prostaglandin H2 synthase isoform 1 (PGHS-1) is the target of the nonsteroidal antiinflammatory drugs (NSAIDs). The X-ray crystal structures of PGHS-1 in complex with the NSAIDs flurbiprofen and bromoaspirin have been determined previously [Picot, D., et al. (1994) Nature 367, 243-249; Loll, P. J., et al. (1995) Nat. Struct. Biol. 2, 637-643]. We report here the preparation and characterization of novel potent iodinated analogs of the NSAIDs indomethacin and suprofen, as well as the refined X-ray crystal structures of their complexes with PGHS-1. The PGHS-iodosuprofen complex structure has been refined at 3.5 A to an R-value of 0.189 and shows the suprofen analog to share a common mode of binding with flurbiprofen. The PGHS-iodoindomethacin complex structure has been refined at 4.5 A to an R-value of 0.254. The low resolution of the iodoindomethacin complex structure precludes detailed modeling of drug-enzyme interactions, but the electron-dense iodine atom of the inhibitor has been unambiguously located, allowing for the placement and approximate orientation of the inhibitor in the enzyme's active site. We have modeled two equally likely binding modes for iodoindomethacin, corresponding to the two principal conformers of the inhibitor. Like flurbiprofen, iodosuprofen and iodoindomethacin bind at the end of the long channel which leads into the enzyme active site. Binding at this site presumably blocks access of substrate to Tyr-385, a residue essential for catalysis. No evidence is seen for significant protein conformational differences between the iodoindomethacin and iodosuprofen of flurbiprofen complex structures.     

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity

BACKGROUND: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis. RESULTS: The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme. CONCLUSIONS: The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.     

Conformational changes of active sites during refolding of urea-denatured creatine kinase

The course of the recovery of the active site conformation during refolding of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. The recovery of the active site conformation has been followed by changes in probe fluorescence of active site of the enzyme labeled by o-phthaldehyde and has been shown to be a triphasic process. A comparison of the rate constants for the conformational recovery of the active site with those for the recovery of enzyme catalytic activity shows that these are synchronized. The results obtained show clearly that, although recovery of partial activity and conformation of the active site occur in reactions with nearly the same rate as those of the refolding processes observed, the complete recovery of the enzyme activity and the active site conformation can only be obtained long after any detectable conformational change of whole enzyme molecule.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting

Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational changes that lead to catalysis. For the free apoenzyme equilibrium favors a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tight and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1559]. It is not clear that binding of either ligand alone is responsible for this conformational switch or whether both are required. In addition, the catalytic subunit binds MgATP and inhibitor peptide synergistically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping the solvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearly distinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensive conformational changes, both local and global, that include the glycine-rich loop, the linker connecting the small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the F helix. These changes, similar to those seen in the ternary complex, are consistent with a transition from an open to a more closed conformation and likely reflect the motions that are associated with catalysis and product release. In contrast, the footprinting pattern of C.PKI resembled free C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, "primes" the enzyme so that it is poised for the docking of PKI and provides an explanation for synergism between MgATP and PKI.     

Inhibitor-induced conformational change in cytochrome P-450CAM

The X-ray crystal structures of cytochrome P-450CAM complexed with both enantiomers of a chiral, multifunctional inhibitor have been refined to R-factors of 21.0% [(+)-enantiomer] and 19.6% [(-)-enantiomer] at approximately 2.1-A resolution. Binding of either enantiomer, both considerably larger than the natural substrate camphor, results in similar, dramatic structural changes in the enzyme. In contrast to all previous P-450CAM crystallographic structures, the Tyr96 side chain is not pointing "down" toward the heme but is rather directed "up" into the proposed substrate access channel. This conformational change is accompanied by the displacement of the Phe193 side chain out into the solvent at the enzyme surface. These changes are consistent with the assignment of this region of the enzyme as the access channel [Poulos et al. (1986) Biochemistry 25, 5314-5322] and suggest that several aromatic residues lining the channel may be involved in substrate recognition and channeling to the active site. The cation usually observed coordinated to the Tyr96 carbonyl oxygen is missing in the presence of the (+)-enantiomer but is present with the (-)-enantiomer. The Phe87 side chain, located near the inhibitor binding site, adopts different orientations depending upon which enantiomer is bound. Finally, electron density reveals that although the inhibitor enantiomers were dichlorinated as provided, when bound to P-450CAM the chlorine atoms are present at only 0-20% occupancy, probably reflecting selective binding of impurities in the samples. Coordinates of these inhibited P-450CAM complexes have been deposited in the Brookhaven Protein Data Bank [Bernstein et al. (1977) J. Mol. Biol. 112, 535-542].