Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.     

Role of conserved Asn-Tyr-Asp-Tyr sequence in bacterial copper/2,4, 5-trihydroxyphenylalanyl quinone-containing histamine oxidase

Copper amine oxidase contains a covalently bound quinonoid cofactor, 2,4,5-trihydroxyphenylalanyl quinone (TPQ), which is synthesized by post-translational modification of a specific tyrosyl residue occurring in the highly conserved sequence, Asn-Tyr-(Asp/Glu)-Tyr. To elucidate the role(s) of the conserved sequence in the biogenesis of TPQ, each of the corresponding residues at positions 401-404 in the recombinant histamine oxidase from Arthrobacter globiformis has been replaced with other amino acids by site-directed mutagenesis. When Asn-401 was changed to Asp or Gln, the rate of TPQ formation by copper-dependent self-processing was 10(3)- to 10(4)-fold slower than in the wild-type enzyme. When Tyr-402 was replaced by Phe, TPQ was not formed at all, showing that Tyr-402 is essential as the precursor to TPQ. In contrast, Asp-403 could be replaced by Glu without changes in the rate of TPQ formation, whereas its replacement by Asn led to a marked decrease. Furthermore, when Tyr-404 was changed to Phe, TPQ was formed swiftly on incubation with copper ions, but the TPQ enzyme exhibited very low activity with altered substrate specificity. These results collectively indicate that a very rigorous structural motif is required for efficient formation of TPQ and for the catalytic activity in the active site of copper amine oxidases.     

Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone

The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.     

Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sci. 2, 1959-1965], while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function [Gong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E. coli strain acquired heme prototrophy. The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits.     

Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.     

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase

Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.     

Characterization of a highly conserved FAD-binding site in human monoamine oxidase B

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.     

His...Asp catalytic dyad of ribonuclease A: structure and function of the wild-type, D121N, and D121A enzymes

The side chains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His...Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His...Asp dyad is composed of His119 and Asp121. Previously, site-directed mutagenesis was used to show that His119 has a fundamental role, to act as an acid during catalysis of RNA cleavage [Thompson, J. E., and Raines, R. T. (1994) J. Am. Chem. Soc. 116, 5467-5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 A with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, Ndelta rather than Odelta of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3'-->5')adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2',3'-cyclic phosphate. Replacing Asp121 decreases the values of kcat/Km and kcat for cleavage by 10-fold (D121N) and 10(2)-fold (D121A). Replacing Asp121 also decreases the values of kcat/Km and kcat for hydrolysis by 10(0. 5)-fold (D121N) and 10-fold (D121A) but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His...Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.     

MutY DNA glycosylase: base release and intermediate complex formation

MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety. We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme. MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage. The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates. The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases. MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY. The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break. Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.     

Conserved residues are functionally distinct within transketolases of different species

Most of the amino acid residues which interact with thiamine pyrophosphate are highly conserved among enzymes which use this cofactor. The possible roles of several such residues in cofactor binding, catalysis, and/or substrate binding were examined for human transketolase. Mutations in H110 resulted in dramatic reductions to 2% or less of the normal activity. No alterations were found in the K(m)app's for the cofactor or for the donor and acceptor substrates. Alterations in Q428 resulted in a less severe loss of activity and also no changes in the K(m)app's. On the basis of the results, H110, an invariant residue, is proposed to function as a base which abstracts a proton from the protonated 4'-iminopyrimidine ring. The deprotonated 4'-imino moiety is required for generation of the C2-thiazolium carbanion which attacks the donor substrate. Interestingly, the function in the human enzyme of this invariant histidine is distinct from its role in yeast transketolase in which it aids in binding donor substrate and in subsequent catalytic events. Q428 is suggested to play a supportive role by stabilizing and orientating a water molecule which mediates the interaction between the 4'-amino group and H110. In other TPP-utilizing enzymes, the equivalent residue of Q428 is a histidine and is thought to deprotonate the 4'-amino group.     

The X-ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 A resolution

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.     

Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase

Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.     

How the substitution of K188 of trypsin binding site by aromatic amino acids can influence the processing of beta-casein

Aspartyl 189 residue of trypsin is known to be essential for specific lysis of Arg-X and Lys-X bonds. Undertaking to modulate the catalytic properties of this protease, otherwise highly conserved K188 was replaced with aromatic amino acid residues aiming the perturbation of the electrostatics and the amplifying of hydrophobic interactions of the substrate binding site. The catalytic properties of the mutants K188F, K188Y, and K188W were measured at pH 7, 8, 9, and 10 using a pair of synthetic tetrapeptide p-nitroanilide substrates and beta-casein. The kinetic analysis reveals that all the mutants conserve the native trypsin capacity to split peptide bonds containing arginyl and lysyl residues. Surprisingly, however, depending on mutation, the optimum pH of activity changes. As demonstrated only by proteolysis of a natural substrate, all mutants cleave also peptide bonds involving asparagine and glutamine. These stuttered cleavage sites are close to the beta-casein fragments in beta-sheet according to Hydrophobic Cluster Analysis.     

Analysis of the site occupancy constraints of primary amino acid sequences in the motif directing palmitylation of the vaccinia virus 37-kDa envelope protein

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1-12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines; A, aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37.     

Analysis of the functional domains of complement receptor type 1 (C3b/C4b receptor; CD35) by substitution mutagenesis

The complement receptor type 1 (CR1; CD35), carrying 30 short consensus repeats (SCRs), has two sites. Site 1 contains SCR-1 and SCR-2 and binds C4b. Site 2 contains SCR-8 and SCR-9 and was reported to bind mainly C3b (Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A., and Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717). For the functional analysis we used two constructs, each with one site. CR1-4, composed of eight and one-half initial SCRs, carries site 1, binds C4b, and is cofactor for C4b cleavage. CR1-4(8,9), obtained from CR1-4 by converting site 1 to site 2, binds iC3/C3b and, unexpectedly, C4b. It is a cofactor for cleavage of both ligands. Its cofactor activity for C4b cleavage is greater than that of site 1. Analysis of the mutants constructed by interchanging homologous peptides between the two sites identified no sequences necessary for cofactor activity other than those required for binding. In site 2, peptides important for both ligands were found. Some modifications of either site led to higher activity for both ligands. Thus the activity of complement regulators can be increased by changing a few amino acids within SCRs, an important step toward the generation of more effective inhibitors of complement activation. Knowledge of the active sites of CR1 should be applicable to other SCR-containing proteins and should provide insights into the evolution of these proteins.     

Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A

Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.     

Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX

VanX, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic Enterococci, is a zinc-containing d-Ala-d-Ala dipeptidase. To identify potential zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue VanX protein. Of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating His116, Asp123, and His184 as zinc-coordinating residues. Homology searches using the 10 amino acid sequence SxHxxGxAxD, in which histidine and aspartate residues are putative zinc ligands, identified the metal coordinating ligands in the N-terminal domain of the murine Sonic hedgehog protein, which also exhibits an architecture for metal coordination identical to that observed in thermolysin from Bacillus thermoproteolyticus. Furthermore, this 10 amino acid consensus sequence is found in the Streptomyces albus G zinc-dependent N-acyl-d-Ala-d-Ala carboxypeptidase, an enzyme catalyzing essentially the same d-Ala-d-Ala dipeptide bond cleavage as VanX, suggesting equivalent mechanisms and zinc catalytic site architectures. VanX residue Glu181 is analogous to the Glu143 catalytic base in B. thermoproteolyticus thermolysin, and the E181A VanX mutant has no detectable dipeptidase activity, yet maintains near-stoichiometric zinc content, a result consistent with the participation of the residue as a catalytic base.     

Monoclonal antibodies against Nalpha-(5'-phosphopyridoxyl)-L-lysine. Screening and spectrum of pyridoxal 5'-phosphate-dependent activities toward amino acids

Cofactors may be expected to expand the range of reactions amenable to antibody-assisted catalysis. The biological importance of pyridoxal 5'-phosphate (PLP) as enzymic cofactor in amino acid metabolism and its catalytic versatility make it an attractive candidate for the generation of cofactor-dependent abzymes. Here we report an efficient procedure to screen antibodies for PLP-dependent catalytic activity and detail the spectrum of catalytic activities found in monoclonal antibodies elicited against Nalpha-(5'-phosphopyridoxyl)-L-lysine. This hapten is a nonplanar analog of the planar, resonance-stabilized coenzyme-substrate adducts formed in the PLP-dependent reactions of amino acids. The hapten-binding antibodies were screened for binding of the planar Schiff base formed from PLP and D- or L-norleucine by competition enzyme-linked immunosorbent assay. The Schiff base (external aldimine) is an obligatory intermediate in all PLP-dependent reactions of amino acids. This simple, yet highly discriminating screening step eliminated most of the total 24 hapten-binding antibodies. Three positive clones bound the Schiff base with L-norleucine, two preferred that with the D-enantiomer. The positive clones were assayed for catalysis of Schiff base formation and of the alpha,beta-elimination reaction with the D- and L-enantiomers of beta-chloroalanine. Three antibodies were found to accelerate aldimine formation, and two of these catalyzed the PLP-dependent alpha,beta-elimination reaction. One of the alpha, beta-elimination-positive antibodies catalyzed the transamination reaction with hydrophobic D-amino acids and oxoacids (Gramatikova, S. I., and Christen, P. (1996) J. Biol. Chem. 271, 30583-30586). All catalytically active antibodies displayed continuous turnover. No PLP-dependent reactions other than aldimine formation, alpha, beta-elimination of beta-chloroalanine and transamination were detected. The successive screening steps plausibly simulate the functional selection pressures having been operative in the molecular evolution of primordial PLP-dependent protein catalysts to reaction- and substrate-specific enzymes.     

Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.