Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.     

Roles of phenylalanine at position 120 and glutamic acid at position 222 in the oxidation of chiral substrates by cytochrome P450 2D6

The roles of Phe-120 and Glu-222 in the oxidation of chiral substrates bunitrolol (BTL) and bufuralol (BF) by CYP2D6 are discussed. Wild-type CYP2D6 (CYP2D6-WT) oxidized BTL to 4-hydroxybunitrolol (4-OH-BTL) with substrate enantioselectivity of (R)-(+)-BTL > (S)-(-)-BTL. The same enzyme converted BF into 1'-hydroxybufuralol with substrate enantioselectivity of (R)-BF > (S)-BF and metabolite diastereoselectivity of (1'R)-OH < (1'S)-OH. The substitution of Phe-120 by alanine markedly increased the apparent K(m) and V(max) values for enantiomeric BTL 4-hydroxylation by CYP2D6. In contrast, the same substitution caused an increase only in V(max) values of (S)-BF 1'-hydroxylation without changing apparent K(m) values, while kinetic parameters (K(m) and V(max) values) for (R)-BF 1'-hydroxylation remained unchanged. Furthermore, the substitution of Glu-222 as well as Glu-216 by alanine remarkably decreased both the apparent K(m) and V(max) values without changing substrate enantioselectivity or metabolite diastereoselectivity. A computer-assisted simulation study using energy minimization and molecular dynamics techniques indicated that the hydrophobic interaction of an aromatic moiety of the substrate with Phe-120 and the ionic interaction of a basic nitrogen atom of the substrate with Glu-222 in combination with Glu-216 play important roles in the binding of BF and BTL by CYP2D6 and the orientation of these substrates in the active-site cavity. This modeling yielded a convincing explanation for the reversal of substrate enantioselectivity in BTL 4-hydroxylation between CYP2D6-WT and CYP2D6-V374M having methionine in place of Val-374, which supports the validity of this modeling.     

Identification of critical residues of choline kinase A2 from Caenorhabditis elegans

Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase.     

Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W., Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M. J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However, pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide [4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with K(s) 1.6 microM. Spirosulfonamide is a substrate for P450 2D6 (k(cat) 6.5 min(-)(1) for the formation of a syn spiromethylene carbinol, K(m) 7 microM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (K(s) 2.5-3.5 microM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on k(cat) but not K(m). Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with K(s) values of 1.7-32 microM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (K(s)) were unknown; these were estimated to be 1.2, 1.5, and 15 microM, respectively (cf. 6 microM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.     

Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J. , Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined. The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pH-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat). Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both. A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M(2+) complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn(2+) at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn(2+), respectively, than the wild type enzyme as detected by 1/T(1) values of water protons, consistent with the loss of a metal ligand. However, the K(m) values of Mg(2+) and Mn(2+) indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex. Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M(2+) complexes. Two-dimensional (1)H-(15)N HSQC and three-dimensional (1)H-(15)N NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in (1)H-(15)N HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region. (1)H-(15)N HSQC titrations of the E53Q, E56Q, and E44D mutants with dGTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site. (ABSTRACT TRUNCATED)     

Crystal structure of human cytochrome P450 2D6

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.     

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.     

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Br?nsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.     

Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding

P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Active site mapping and substrate channeling in the sterol methyltransferase pathway

Sterol methyltransferase (SMT) from Saccharomyces cerevisiae was purified from Escherichia coli BL21(DE3) and labeled with the mechanism-based irreversible inhibitor [3-3H]26,27-dehydrozymosterol (26,27-DHZ). A 5-kDa tryptic digest peptide fragment containing six acidic residues at positions Glu-64, Asp-65, Glu-68, Asp-79, Glu-82, and Glu-98 was determined to contain the substrate analog covalently attached to Glu-68 by Edman sequencing and radioanalysis using C18 reverse phase high performance liquid chromatography. Site-directed mutagenesis of the six acidic residues to leucine followed by activity assay of the purified mutants confirmed Glu-68 as the only residue to participate in affinity labeling. Equilibration studies indicated that zymosterol and 26,27-DHZ were bound to native and the E68L mutant with similar affinity, whereas S-adenosylmethionine was bound only to the native SMT, K(d) of about 2 microm. Analysis of the incubation products of the wild-type and six leucine mutants by GC-MS demonstrated that zymosterol was converted to fecosterol, 26,27-DHZ was converted to 26-homo-cholesta-8(9),23(24)E,26(26')-trienol as well as 26-homocholesta-8(9),26(26')-3beta,24beta-dienol, and in the case of D79L and E82L mutants, zymosterol was also converted to a new product, 24-methylzymosta-8,25(27)-dienol. The structures of the methylenecyclopropane ring-opened olefins were determined unambiguously by a combination of (1)H and (13)C NMR techniques. A K(m) of 15 microm, K(cat) of 8 x 10(-4) s(-1), and partition ratio of 0.03 was established for 26,27-DHZ, suggesting that the methylenecyclopropane can serve as a lead structure for a new class of antifungal agents. Taken together, partitioning that leads to loss of enzyme function is the result of 26,27-DHZ catalysis forming a highly reactive cationic intermediate that interacts with the enzyme in a region normally not occupied by the zymosterol high energy intermediate as a consequence of less than perfect control. Alternatively, the gain in enzyme function resulting from the production of a delta(25(27))-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.     

Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity

The catalytic reaction mechanism and binding of substrates was investigated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mechanism, in which E52 acts as an initiating base and R105 is thought to stabilize the transition state of the reaction. Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased k(cat), while the catalytic activity of E52Q and R105K was abolished. The E52D mutant was crystallized with its feedback inhibitor dTTP. The backbone conformation remained unchanged, and coordination between D52 and the dTTP-Mg complex was observed. The observed decrease in k(cat) for E52D was most likely due to an increased distance between the catalytic carboxyl group and 5'-OH of deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N and Y70 to W was carried out to investigate substrate binding. The mutations primarily affected the K(m) values, whereas the k(cat) values were of the same magnitude as for the wild-type. The Y70W mutation made the enzyme lose activity towards purines and negative cooperativity towards dThd and dCyd was observed. The Q81N mutation showed a 200- and 100-fold increase in K(m), whereas k(cat) was decreased five- and twofold for dThd and dCyd, respectively, supporting a role in substrate binding. These observations give insight into the mechanisms of substrate binding and catalysis, which is important for developing novel suicide genes and drugs for use in gene therapy.     

Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases.

The full-length and ectodomain forms of beta-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the beta-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2'0 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM/DAEFR, K(m) = 7 microm, K(cat) = 0.002 s(-1); Swedish mutant, SEVNL/DAEFR, K(m) = 9 microm, K(cat) = 0.02 s(-1)). A new substrate (VVEVDA/AVTP, K(m) = 1 microm, K(cat) = 0.004) was identified by serendipity.     

Intermediate trapping on a mutant retaining alpha-galactosyltransferase identifies an unexpected aspartate residue

Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.     

tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-(14)C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations. The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.     

Role of Asp-9 and Glu-36 in the active site of the pneumococcal CPL1 lysozyme: an evolutionary perspective of lysozyme mechanism.

The role of carboxylic amino acids Asp-9 and Glu-36 in the activity of CPL1 lysozyme was investigated by site-directed mutagenesis. The enzymatic activity of the single mutants D9E, D9N, D9H, D9K, D9A, E36D, E36Q, E36K, and E36A and of the double mutant D9A-E36A was analyzed using a highly sensitive radioactive assay. All mutants but D6K showed detectable activities. Interestingly, the mutants E36D and E36Q retained 67% and 37% activity, respectively. Amino acid replacements at position 9 turned out to be more critical for activity than at position 36. In analogy to the mechanism described for hen egg-white lysozyme, where the proton donor play a central role, we propose that, in the CPL1 lysozyme, Asp-9 might act as the proton donor for activation of the substrate, and Glu-36 could help in the stabilization of the intermediate oxocarbocation. The residual activity of lysozyme mutants lacking one or two of the acidic amino acids may be explained by the participation of a water molecule as proton donor and/or to electrostatic contributions in the active center stabilizing the transition state of the reaction. Our results are in agreement with the hypothesis that enzymes have been optimized during evolution from an ancestral protein able to bind more tightly the transition state of the substrate than the substrate itself, by the acquisition of amino acids serving a function in catalysis.     

Structure of 6-oxo camphor hydrolase H122A mutant bound to its natural product, (2S,4S)-alpha-campholinic acid: mutant structure suggests an atypical mode of transition state binding for a crotonase homolog

The crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic beta-diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744-1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of k(cat)/K(m) derived from a very large increase in K(m) for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of k(cat), and its K(m) is five times that of the wild-type. The location of the active site is confirmed by the 1.9-A structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)-alpha-campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members.     

Giardia lamblia RNA cap guanine-N2 methyltransferase (Tgs2)

Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine cap structures of small nuclear and small nucleolar RNAs. Whereas budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe encode a single Tgs1 protein, the primitive eukaryote Giardia lamblia encodes two paralogs, Tgs1 and Tgs2. Here we show that purified Tgs2 is a monomeric enzyme that catalyzes methyl transfer from AdoMet (K(m) of 6 microm) to m(7)GDP (K(m) of 65 microm; k(cat) of 14 min(-1)) to form m(2,7)GDP. Tgs2 also methylates m(7)GTP (K(m) of 30 microm; k(cat) of 13 min(-1)) and m(7)GpppA (K(m) of 7 microm; k(cat)) of 14 min(-1) but is unreactive with GDP, GTP, GpppA, ATP, CTP, or UTP. We find that the conserved residues Asp-68, Glu-91, and Trp-143 are essential for Tgs2 methyltransferase activity in vitro. The m(2,7)GDP product formed by Tgs2 can be converted to m(2,2,7)GDP by S. pombe Tgs1 in the presence of excess AdoMet. However, Giardia Tgs2 itself is apparently unable to add a second methyl group at guanine-N2. This result implies that 2,2,7-trimethylguanosine caps in Giardia are either synthesized by Tgs1 alone or by the sequential action of Tgs2 and Tgs1. The specificity of Tgs2 raises the prospect that some Giardia mRNAs might contain dimethylguanosine caps.     

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.