The Copper-Enzyme Family of Dopamine β-Monooxygenase and Peptidylglycine α-Hydroxylating Monooxygenase: Resolving the Chemical Pathway for Substrate Hydroxylation

Dopamine β-monooxygenase (DβM) 2The abbreviations used are: DβM, dopamine β-monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase; DFPEA, β,β-difluorophenethylamine. 2The abbreviations used are: DβM, dopamine β-monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase; DFPEA, β,β-difluorophenethylamine. and peptidylglycine α-hydroxylating monooxygenase (PHM) belong to a small class of copper proteins found exclusively in higher eukaryotes. These physiologically important enzymes catalyze the transformation of dopamine to norepinephrine (DβM) (Equation 1) and C-terminal glycine-extended peptides to their α-hydroxylated products (PHM) (Equation 2). Although their substrate specificities are grossly different, these enzymes greatly resemble each other in many other respects.EQUATION 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both enzymes are localized in subcellular compartments: the chromaffin vesicles of the adrenal gland or synaptic vesicles of the sympathetic nervous system (DβM) (1Stewart L.C. Klinman J.P. Annu. Rev. Biochem. 1988; 57: 551-591Crossref PubMed Google Scholar) and the secretory vesicles of the pituitary gland (PHM) (2Eipper B.A. Stoffers P.A. Mains R.E. Annu. Rev. Neurosci. 1992; 15: 57-85Crossref PubMed Scopus (559) Google Scholar). Although DβM and PHM exist in soluble and membrane-bound forms within their vesicular compartments (3Winkler H. Carmichael S.W. Poisner A.M. Trifaro J.M. The Secretory Granule. Elsevier Biomedical Press, Amsterdam1982Google Scholar, 4Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (373) Google Scholar), the majority of mechanistic studies have been completed with the more tractable soluble enzymes. The physiological roles played by soluble and membrane-bound enzymes may be different, but the chemical mechanisms are almost certain to be the same. The in vivo production of PHM is accompanied by the expression of a second activity that leads to the cleavage of the α-hydroxypeptide in Equation 2 to the biologically active, C-terminally carboxamidated peptide and glyoxylate (5Kulathila R. Merkler K.A. Merkler D.J. Nat. Prod. Rep. 1999; 16: 145-154Crossref PubMed Scopus (89) Google Scholar). This second activity is found either in a separate enzyme (peptidylglycine α-amidating lyase) or within a single polypeptide chain that also contains the activity of Equation 2 (peptidylglycine α-amidating monooxygenase). The in vitro study of the hydroxylating activity (Equation 2) is commonly performed with a construct in which the first 41 amino acids of PHM have been deleted, referred to as the PHM catalytic core (PHMcc). Comparison of the primary sequence of PHMcc with the larger DβM indicates a central core of ∼300 amino acids from DβM that is 27% identical and 40% homologous to PHM (6Lamoroux A. Vigny A. Faucon Biguet V. Darmon M.C. Frank R. Henry J.P. Mallet J. EMBO J. 1987; 6: 3931-3937Crossref PubMed Scopus (94) Google Scholar, 7Southan C. Kruse L.I. FEBS Lett. 1989; 255: 116-120Crossref PubMed Scopus (68) Google Scholar, 23Eipper B.A. Quon A.S.W. Mains R.E. Boswell J.S. Blackburn N.J. Biochemistry. 1995; 34: 2857-2865Crossref PubMed Scopus (102) Google Scholar). In addition, DβM contains ∼200 amino acids toward its N terminus and another ∼100 amino acids toward the C terminus that bear no relationship to PHM. Of particular note is the conservation of the ligands to the two coppers per enzyme subunit, designated as CuH and CuM. Using the numbering system of PHM, these are His-107, His-108, and His-172 for the CuH site and His-242, His-244, and Met-314 for the CuM site. Beginning in l997, a series of x-ray structures of different forms of PHM were published. The representation in Fig. 1 illustrates the disposition of the two copper centers per subunit and their respective ligands (8Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar). Additionally, a substrate analog, N-acetyl, diiodo-Tyr-Gly, is shown to bind close to the CuM site, adjacent to Arg-240, implicating the CM domain as the site of substrate hydroxylation. A recent x-ray structure of PHM with a slowly reacting substrate bound at the CuM site shows the presence of a bound O2 in close proximity to substrate as well (9Prigge S.T. Eipper B.A. Mains R.E. Amzel L.M. Science. 2004; 304: 864-867Crossref PubMed Scopus (358) Google Scholar). The structure of PHM reveals many striking and unexpected features. These include a lack of bridging ligands between the copper sites, consistent with earlier EPR studies that had failed to show any spin coupling between the paramagnetic copper centers (10Klinman J.P. Chem. Rev. 1996; 96: 2541-2561Crossref PubMed Scopus (800) Google Scholar). It is known that the two electrons consumed during substrate hydroxylation are stored in the CuH and CuM sites (11Brenner M. Klinman J.P. Biochemistry. 1989; 28: 4664-4670Crossref PubMed Scopus (70) Google Scholar). Although it was conceivable that the metal centers could approach each other during the catalytic cycle, there is no structural evidence for a hinge region capable of facilitating such a movement. Although DβM and PHM belong to a multi-copper family of proteins, the coppers appear to perform different functions, that of substrate hydroxylation (CuM) and electron storage/transfer (CuH). Perhaps the most startling feature to emerge from x-ray studies is the fully solvent-exposed nature of the copper sites, raising the questions of (i) how DβM and PHM carry out the regio- and stereospecific hydroxylations of Equations 1 and 2 and (ii) how they carry out controlled electron transfer from CuH to CuM through bulk solvent. A successful, high level expression system has never been established for DβM, precluding detailed x-ray structural studies and site-specific mutagenesis studies. However, many similar kinetic and mechanistic probes have been applied to DβM and PHM, indicating an extraordinary conservation of properties. Both enzymes have been shown to undergo a cyclical reduction of their copper sites by ascorbic acid, the physiologically relevant and preferred reductant (11Brenner M. Klinman J.P. Biochemistry. 1989; 28: 4664-4670Crossref PubMed Scopus (70) Google Scholar, 12Freeman J.C. Villafranca J.J. Merkler D.J. J. Am. Chem. Soc. 1993; 115: 4923-4924Crossref Scopus (49) Google Scholar). The resulting Cu(I) sites are returned to the +2 valence state in the presence of substrate and dioxygen via a formal “ping-pong” mechanism in which reductant and substrates interact with different forms of enzyme that are separated by irreversible chemical processes. A ping-pong mechanism indicates that both electrons needed for the hydroxylation of substrate can be stored on the enzyme. In support of such a mechanism, single turnover freeze or acid quench studies of reduced DβM in the absence of exogenous reductant have demonstrated concomitant formation of Cu(II) and hydroxylated product in kinetically competent processes (11Brenner M. Klinman J.P. Biochemistry. 1989; 28: 4664-4670Crossref PubMed Scopus (70) Google Scholar, 13Brenner M. Murray C.J. Klinman J.P. Biochemistry. 1989; 28: 4656-4664Crossref PubMed Scopus (40) Google Scholar). These properties require that the electron stored on CuH be capable of transferring to the CuM site at a rate compatible with catalytic turnover. One of the major goals of investigation of DβM and PHM has been to isolate their chemical steps, such that detailed mechanistic information can be inferred regarding the nature of O2 and substrate activation. Use of deuterated substrates of comparable reactivity (dopamine and hippuric acid with DβM and PHM, respectively) leads to relatively small kinetic isotope effects, indicating that substrate activation is only partially rate determining under steady state conditions. However, comparison of experimentally determined deuterium and tritium isotope effects has permitted calculation of the magnitude of deuterium isotope effects on the isolated C–H cleavage step (14Francisco W. Merkler D. Blackburn N. Klinman J.P. Biochemistry. 1998; 37: 8244-8252Crossref PubMed Scopus (83) Google Scholar, 15Miller S.M. Klinman J.P. Biochemistry. 1982; 22: 3091-3096Crossref Scopus (60) Google Scholar, 16Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar). These “intrinsic” values for kH/kD (Table 1) show remarkable agreement between DβM and PHM; despite their differing sequences and substrate specificities, both enzymes catalyze C–H abstraction reactions via identical activated complexes! These results reinforce the assumption, based on sequence comparisons, that DβM and PHM function via identical chemical mechanisms.TABLE 1PHM and DβM proceed by identical transition states (Refs. 14Francisco W. Merkler D. Blackburn N. Klinman J.P. Biochemistry. 1998; 37: 8244-8252Crossref PubMed Scopus (83) Google Scholar, 15Miller S.M. Klinman J.P. Biochemistry. 1982; 22: 3091-3096Crossref Scopus (60) Google Scholar, and 17Tian G. Berry J.A. Klinman J.P. Biochemistry. 1994; 33: 226-234Crossref PubMed Scopus (118) Google Scholar)IntrinsicObservedEnzymekH/kD for C—Hk16/k18 for O—O1°2°C—HC—DDβMaSubstrate for DβM is dopamine: kC-H = 1200 s-1; bond dissociation energy = 85 kcal/mol.10.91.191.01971.0256(1.9)(0.06)(0.0003)(0.0003)PHMbSubstrate for PHM is hippuric acid: kC-H = 810 s-1; bond dissociation energy = 87 kcal/mol.10.61.201.01671.0216(0.8)(0.03)(0.0032)(0.0014)a Substrate for DβM is dopamine: kC-H = 1200 s-1; bond dissociation energy = 85 kcal/mol.b Substrate for PHM is hippuric acid: kC-H = 810 s-1; bond dissociation energy = 87 kcal/mol. Open table in a new tab Also shown in Table 1 are the magnitudes of observed O-18 kinetic isotope effects using either H- or D-labeled substrates (14Francisco W. Merkler D. Blackburn N. Klinman J.P. Biochemistry. 1998; 37: 8244-8252Crossref PubMed Scopus (83) Google Scholar, 17Tian G. Berry J.A. Klinman J.P. Biochemistry. 1994; 33: 226-234Crossref PubMed Scopus (118) Google Scholar). Although the measured values differ somewhat between DβM and PHM, consistent with somewhat different rate-limiting steps under steady state conditions, in both instances the magnitude of k16/k18 is seen to increase when substrate is deuterated. This highly significant observation demonstrates that the activation of both substrate and O2 must be fully connected by a reversible chemical process. Hydrogen Transfer Is Non-classical—With the inescapable connection between CH and O2 activation in monooxygenase reactions, early studies focused on characterizing the properties of CH activation. Structure reactivity correlations on DβM, using a series of ring-substituted phenethylamines, indicated a slope of +1.5, i.e. a significantly faster rate with ring-donating substituents (15Miller S.M. Klinman J.P. Biochemistry. 1982; 22: 3091-3096Crossref Scopus (60) Google Scholar). This was originally interpreted in terms of an electron-deficient transition state resulting from hydrogen atom transfer between substrate and activated O2. However, as elaborated below, the H-transfer reaction has been reformulated in the context of a modified Marcus model, whereby hydrogen transfer occurs quantum mechanically and trends in reactivity are expected to reflect the impact of reaction driving force on rate (cf. Ref. 18Knapp M.J. Rickert K. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 3865-3874Crossref PubMed Scopus (432) Google Scholar). Four possible mechanisms for O2 activation in DβM and PHM can be outlined for experimental testing. These range from the formation of a one-electron reduced intermediate (metal superoxo, CuM(II) O2−˙), Mechanism I, to two-electron reduced species (metal peroxo, CuM(II)(O22−)), Mechanism II, or metal hydroperoxo (CuM(II) (HO2−)), Mechanism III, and finally to a highly reduced CuM(II) (O·¯) formed via the reductive cleavage of CuM(II) (HO2−) by a conserved active site tyrosine, Mechanism IV. One of the most puzzling observations on DβM came from measurements of O-18 discrimination in the uptake of O2 using phenethylamine substrates. At the time of these measurements, it was generally assumed that a peroxo-type species, which had accepted one electron each from both CuM and CuH, would be the functional oxidizing species (Mechanism II or III). With the expectation that there would be more bond cleavage at the OO peroxo bond as the reaction became less favorable (later transition state), the O-18 isotope effect should have become larger with decreasing substrate reactivity. The experimental data indicated a decreasing magnitude for the O-18 kinetic isotope effect as the rate constant for CH activation fell over 2.5 orders of magnitude (18Knapp M.J. Rickert K. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 3865-3874Crossref PubMed Scopus (432) Google Scholar). In an effort to reconcile the conflict between observation and prediction, a mechanism was put forth in which a copper hydroperoxo species reacted further with a conserved active site tyrosine near CuM to yield the copper oxo species as the reactive intermediate (Mechanism IV). Although this mechanism was not easily tested with DβM, eventual preparation of the tyrosine mutant of PHM, Y318F, indicated no impact on kcat and, most significantly, less than a 4-fold reduction in the rate of CH activation. This site-specific mutagenesis experiment effectively eliminated Mechanism IV from consideration (19Francisco W.A. Blackburn N.J. Klinman J.P. Biochemistry. 2003; 42: 1813-1819Crossref PubMed Scopus (64) Google Scholar). The lack of agreement between experimental and predicted trends in O-18 isotope effects argued for a reassessment of underlying assumptions. Given the growing evidence for H-tunneling in enzyme systems, the most reasonable explanation seemed to lie with the presence of non-classical behavior in PHM and DβM; this would preclude a simple prediction of the “position of the H-transfer transition state” from trends in substrate reactivity. In fact, early evidence for “deviant behavior” in the DβM and PHM reactions had been evident from the comparison of the magnitudes of intrinsic primary and secondary hydrogen isotope effects (Table 1). Kinetic isotope effects that are formulated in the context of transition state theory are expected to yield similar conclusions about the structure of the transition state, using either intrinsic primary or secondary effects. Yet, the data in Table 1 implicate a symmetrical transition state (from the l° kinetic isotope effect) versus a late transition state (from the 2° kinetic isotope effect). Definitive evidence for non-classical behavior in DβM and PHM became available from a detailed investigation of hydrogen isotope effects as a function of temperature. Recognizing that chemistry is unlikely to be rate-determining under the available experimental conditions, Francisco et al. (16Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar) pursued methods for the very precise determination of primary deuterium and tritium isotope effects. From the measured isotope effects, the intrinsic primary hydrogen isotope effect on the isolated CH cleavage step could be analyzed as a function of temperature, indicating little change across the experimental temperature range (Ea(D) ≃ Ea(H))(16Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar). By contrast, the semi-classical theory of isotope effects that incorporates the zero point vibrational energy of the reacting bond predicts that the isotope effect will depend on temperature and disappear in the high temperature regime. Numerous enzyme systems have now been documented to show a similar of isotope effects, implicating a quantum transfer of hydrogen that is to the J.P. Google Scholar, M.J. Klinman J.P. J. Biochem. 2002; PubMed Scopus Google Scholar). the for the is of that the data for PHM are well using a modified (16Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar). In the of the temperature behavior of the isotope effects, it was to the experimentally observed of activation steps that activation to hydrogen Given the that these steps be the most was that activated is formed in an by a second rate-limiting The driving force for formation of from O2 that the (Mechanism may not be a for the activated O2 species in DβM and PHM. an experimental approach to for the presence of it was that formation of at the highly solvent-exposed active site of would to of in the presence of substrates of greatly reduced intrinsic reactivity. This was with DβM, using substrates that by 2 in reactivity. in Ref. J.P. K. Klinman J.P. J. Biol. Chem. 2003; PubMed Scopus Google Scholar, of the of O2 to product formation indicated coupling under conditions. using PHM and a mutant at the CuH site that its to bind copper almost no in the of coupling between O2 and substrate was N. J. and J. in This behavior is in to other enzymes known to metal of O2 reduction and substrate hydroxylation are observations (cf. N. R. J. Chem. Rev. 2000; PubMed Scopus Google and M.J. J. Biol. Chem. PubMed Google Scholar). In to a metal from the available data also appear to out a mechanism in which O2 was to bind to CuH to a by dissociation of the and its across the to bind and be further reduced at CuM Blackburn N.J. Biochemistry. 1999; PubMed Scopus (70) Google Scholar). to a of of O2 uptake and substrate would be expected for a mechanism of this now to the as an species, Mechanism II, in the absence of a the may be expected to be bound at CuM. Although early kinetic studies on DβM had toward a for acid in O2 the x-ray structure of PHM failed to an active site functional capable of such This to the of a copper as the catalytic species (8Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar). One feature of CuM(II) is the prediction that both coppers will undergo in the of O2 reduction to CuM(II) the copper centers are expected to at a in the intermediate and, it should be possible to the Cu(II) EPR in a this a of was and reacted with DβM in a J.P. K. Klinman J.P. J. Biol. Chem. 2003; PubMed Scopus Google Scholar). a phenethylamine substrate that leads to a formation of Cu(II) that with product little of the copper centers was observed with the on the time of the experiment J.P. K. Klinman J.P. J. Biol. Chem. 2003; PubMed Scopus Google Scholar). Although it is to out of as well as the formation of such a level of CuM(II) that it is of these is very A the formation of an EPR species in the presence of DFPEA, to very Mechanism I, the formation of a to a mechanism that is capable of the of data available for DβM and PHM. The mechanism of Fig. 2 also an to the of how these enzymes catalyze electron transfer across bulk at a rate that is compatible with catalytic turnover. with the fully reduced enzyme on the of Fig. substrate and O2 bind to the This is the for O2 activation electron transfer from to O2 to the EPR The is to be in an consistent with the impact of substrate on the O-18 isotope effects and the that uptake and product formation are functional theory support such an of CuM(II) in J. Am. Chem. Soc. 2004; PubMed Scopus Google Scholar). the active sites, the reactivity of the species is expected to be to the of transfer from metal to with the of of the to transfer of a hydrogen atom from substrate via will also be to the of the to many different only a of these is expected to the and that can to function from the hydrogen in the substrate to (cf. Ref. M.J. Klinman J.P. J. Biochem. 2002; PubMed Scopus Google Scholar). These properties that a of of is at within the active sites of DβM and PHM. One of the more of the mechanism in Fig. 2 the at which the second electron from the reaction The rate constant for the CH bond cleavage step with the substrate dopamine in DβM is almost (16Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar). electron transfer from CuH to CuM the substrate activation it would have to significantly faster than have how the electron could this through bulk that of the the substrate J. R. D. Mains R.E. Eipper B.A. Biochemistry. 2003; 42: PubMed Scopus Google and W.A. G. Merkler D.J. Klinman J.P. J. Am. Chem. Soc. 2004; PubMed Scopus Google Scholar). of these failed to support for their In the context of Fig. the second electron not transfer to the CuM site an irreversible hydrogen atom the range electron transfer which is a J. R. D. Mains R.E. Eipper B.A. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). This not only the kinetic for the electron transfer step but its driving In the mechanism for DβM by et al. J.P. K. Klinman J.P. J. Biol. Chem. 2003; PubMed Scopus Google Scholar), the intermediate is to undergo reductive cleavage by CuH via to and a which with the to an The of and J. Am. Chem. Soc. 2004; PubMed Scopus Google Scholar) an in which the from the to the resulting species reduction by CuH to in a favorable One feature of the DβM mechanism that may the product shown in Fig. 2 is the observation that kcat is faster with phenethylamine substrates This has been used to for the of an product that rate-limiting dissociation to product as of the kcat (15Miller S.M. Klinman J.P. Biochemistry. 1982; 22: 3091-3096Crossref Scopus (60) Google Scholar). The of the systems a in which the and of early studies were by such as the reactivity of metal Chem. PubMed Scopus Google Scholar) or chemistry M. Chem. Rev. 1996; 96: PubMed Scopus Google Scholar). have a new these and enzymes have to their experimental which include the precise of the active site for hydrogen transfer and the possible of of the from the active site in this process. The of the mechanism of range electron transfer between the CuH and CuM sites also for and and the and have their energy and to these studies.