The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase (PHM): a spectroscopic study of the H172A mutant

The spectroscopic characterization of the H172A mutant of peptidylglycine alpha-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu(H) ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 A in wild-type PHM to 1.89 A in H172A associated with a change in the structure of Cu(H) from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu(M) site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu(H) was obtained through the observation that loss of the H172 Cu(H) ligand caused a 3 cm(-)(1) blue shift in the nu(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.     

The catalytic copper of peptidylglycine alpha-hydroxylating monooxygenase also plays a critical structural role

Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.     

Genomic organization and splicing variants of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

Cnidarians are primitive animals that use neuropeptides as their transmitters. All the numerous cnidarian neuropeptides isolated, so far, have a carboxy-terminal amide group that is essential for their actions. This strongly suggests that alpha-amidating enzymes are essential for the functioning of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), which is coded for by a single gene. In a previous paper (F. Hauser et al., Biochem. Biophys. Res. Commun. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from the sea anemone Calliactis parasitica. In the present paper we have determined the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 exons and 14 introns. The last coding exon (exon 15) contains a stop codon, leaving no room for PAL and, thereby, for a bifunctional PAM enzyme as in mammals. Furthermore, we found a CP1 splicing variant (CP1-B) that contains exon-9 instead of exon-8, which was present in the previously characterized PHM cDNA (CP1-A). CP1-A and -B have 97% amino acid sequence identity, whereas both splicing variants have around 42% sequence identity with the PHM part of rat PAM. Essential amino acid residues for the catalytic activity and the 3D structure of PHM are conserved between CP1-A, -B and the PHM part of rat PAM. Furthermore, eight introns in CP1 occur in the same positions and have the same intron phasing as eight introns in the rat PAM gene, showing that the sea anemone PHM is not only structurally, but also evolutionarily related to the PHM part of rat PAM.     

Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine alpha-hydroxylating monooxygenase: mechanistic implications

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) and dopamine beta-monooxygenase (DbetaM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation of peptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby these enzymes activate molecular oxygen and the C-H bond of substrate has been the subject of numerous studies, and various mechanisms have been put forth. From the magnitude of (18)O isotope effects as a function of substrate structure in DbetaM, an active site tyrosine had been proposed to function in the reductive activation of Cu(II)-OOH to generate a reactive copper-oxo species [Tian et al. (1994) Biochemistry 33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmed from crystallographic studies [Prigge et al. (1997) Science 278, 1300]. We now report extensive kinetic and isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in the catalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and (18)O isotope effects that are within experimental error of the wild-type enzyme and that the mutation causes only a slight reduction in the rate constant for C-H bond cleavage. These findings, together with the recent demonstration that C-H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J. Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class of copper enzymes.     

Characterization of a Xenopus laevis skin peptidylglycine alpha-hydroxylating monooxygenase expressed in insect-cell culture.

The C-terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two-step reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase. A Xenopus laevis PHM expressed in insect-cell culture by the baculovirus-expression-vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L-ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L-ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping-pong mechanism with respect to peptide and L-ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C-termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non-polar amino acids.     

Major changes in copper coordination accompany reduction of peptidylglycine monooxygenase: implications for electron transfer and the catalytic mechanism

H and Cu_M). The Cu_H center changes from 4- or 5-coordinate tetragonal to a 2-coordinate configuration, with one of the three histidine ligands becoming undetectable by EXAFS (suggesting that it has moved away from the Cu_H by at least 0.3 Å). The Cu_M center changes from 4- or 5-coordinate tetragonal to a trigonal or tetrahedral configuration, with an estimated 0.3–0.5 Å movement of the M314 S ligand. Reduction also leads to loss of coordinated water from both of the coppers. Substrate binding has little or no effect on the local environment of the Cu centers in either oxidation state. These findings bring into question whether direct electron transfer between Cu_H and Cu_M via a tunneling mechanism can be fast enough to support the observed catalytic rate, and suggest that some other mechanism for electron transfer, such as superoxide channeling, should be considered. Received: 17 November 1999 / Accepted: 25 February 2000     

Isocyanide binding to the copper(I) centers of the catalytic core of peptidylglycine monooxygenase (PHMcc)

Binding of the Cu(I)-specific ligands 2,6-dimethylphenyl isocyanide (DIMPI) and isopropyl isocyanide (IPI) to the reduced form of peptidylglycine monooxygenase (PHM) is reported. Both ligands bind to the methionine-containing CuM center, eliciting FTIR bands at 2,138 and 2,174 cm(-1), respectively, but appear unable to coordinate at the histidine-containing CuH center in the wild-type enzyme. This chemistry parallels that previously observed for CO binding to the reduced PHM catalytic core (PHMcc). However, in contrast to the CO chemistry, peptide substrate binding did not induce binding of the isocyanide at CuH. XAS confirmed the binding of DIMPI at CuM via the observation of a short Cu-C interaction at 1.87 A and by the lengthening of the Cu-S(methionine) bond length by 0.06 A. Similarly, FTIR studies on DIMPI binding to the M314I and H172A mutant forms of reduced PHMcc confirmed the assignment of the 2,138-cm(-1) IR band as a CuM-DIMPI complex, but surprisingly also showed DIMPI binding to CuH, as indicated by a band at 2,148 cm(-1). An inorganic complex, [Cu(1,2-Me2Im)2(DIMPI)](PF6), was synthesized and its crystal structure was determined as a model for the interaction of isocyanides with imidazole-containing Cu(I) complexes. Comparison of EXAFS data for the protein and model suggests that DIMPI probably binds to CuM in a tilted fashion, similar to that of ethyl isocyanide binding to myoglobin.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.     

Large scale production of the copper enzyme peptidylglycine monooxygenase using an automated bioreactor

Rat PHM (peptidylglycine alpha-hydroxylating monooxygenase; EC 1.14.17.3) expressed in CHO DG44 cells as a recombinant protein (rat PHMcc, residues 42-356 cloned in the pCIS vector, A.S. Kolhekar, H.T. Keutman, R. E. Mains, A.S.W. Quon, B.A. Eipper, Biochemistry 36 (1997) 10901-10909), was produced in two different bioreactors, a Cellmax 100 (B1) and an Accusyst-MiniMax (B2). B2 contains features not present in B1, which contribute to environmental control, and ease of operation, and was more successful at producing high quality PHM than B1 in both yield (B1: 5mg/day, B2: 12-15 mg/day), activity (B1: 12-20 micromol O(2)/min/mg, B2: 24-36 micromol O(2)/min/mg), and viability (B1: <6 months, B2: indefinite). Additionally, B1 exhibited clipping at Ser 61, and a decline in quality late in the run. PHM from B2 was of consistent quality and homogeneity throughout the run. The increased yield and purity made possible collection of visible spectra of the Cu(II) sites, and mass spectrometric data not previously available.     

A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK(a) of 4.6 to generate the inactive low-pH form with Cu(H) coordinated by M109, H107, and H172.     

Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper

A derivative of peptidylglycine monooxygenase which lacks the CuH center has been prepared and characterized. This form of the enzyme is termed the half-apo protein. Copper-to-protein stoichiometric measurements establish that the protein binds only one of the two copper centers (CuM and CuH) found in the native enzyme. Confirmation that the methionine-containing CuM has been retained has been obtained from EXAFS experiments which show that the characteristic signature of the Cu-S(Met) interaction is preserved. The half-apo derivative binds 1 equiv of CO per copper with an IR frequency of 2092 cm(-1), and this monocarbonyl also displays the Cu-S(Met) interaction in its EXAFS spectrum. These results allow unambiguous assignment of the 2092 cm(-1) band as a CuM-CO species. Binding of CO in the presence of peptide substrate was also investigated. In the native enzyme, substrate induced binding of a second CO molecule with an IR frequency of 2062 cm(-1), tentatively assigned to a CO complex of the histidine-containing CuH site. Unexpectedly, this reactivity is also observed in the half-apo derivative, although the intensity distribution of the CO stretches now indicates that the copper has been partially transferred to a second site, believed to be CuH. The implications of this observation are discussed in terms of a possible additional peptide binding site close to the CuH center.     

The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase

The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.     

The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.     

Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study

Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction—the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O₂-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O₂ is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (Cu_H and Cu_M). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-Å-resolution structure (hydro)peroxide binds solely to Cu_M in a slightly asymmetric side-on mode. The O–O interatomic distance of the copper-bound ligand is 1.5 Å, characteristic of peroxide/hydroperoxide species, and the Cu–O distances are 2.0 and 2.1 Å. Density functional theory calculations using the first coordination sphere of the Cu_M active site as a model system show that the computed energies of the side-on L₃Cu_M(II)–O₂ ^2? species and its isomeric, end-on structure L₃Cu_M(I)–O₂ ^·? are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L₃Cu_M(II)O₂ ^2? is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.     

Structural and kinetic analyses of the H121A mutant of cholesterol oxidase

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a approximately 100 mV decrease in the midpoint redox potential and a approximately 40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024-18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 A resolution (1 A identical with 0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180 degrees about chi1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.     

The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A

We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.     

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.