Trihydroxyphenylalanine quinone (TPQ) from copper amine oxidases and lysyl tyrosylquinone (LTQ) from lysyl oxidase

TRIHYDROXYPHENYLALANINE QUINONE (TPQ) FROM COPPER AMINE OXIDASES AND LYSYL TYROSYLQUINONE (LTQ) FROM LYSYL OXIDASE BY JOANNE E. DOVE AND JUDITH P. KLINMAN Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Topa Quinone (TPQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Copper Amine Oxidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Enzyme Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. TPQ Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Roles of Conserved Active Site Residues in Catalysis and Biogenesis . . . . . E. Biological Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Lvsine Tyrosylquinone (LTQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . "v~ I~ysylOxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, Enzwne Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B, Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (;. LTQ Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Biological Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Perspective and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ret~'rences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 143 143 143 147 154 159 162 [64 165 165 t 61) 1(~8 169 170 172

I. INTRODUCTION

P o s t t r a n s l a t i o n a l m o d i f i c a t i o n o f a r o m a t i c a m i n o side c h a i n s is n o w a well-established m e a n s o f i n t r o d u c i n g n e w f u n c t i o n a l i t i e s i n t o e n z y m e active sites. T h e d e m o n s t r a t i o n o f a tyrosyl radical in t h e r i b o n u c l e o t i d e r e d u c t a s e s was t h e first e x a m p l e o f a m o d i f i e d tyrosine side c h a i n in a p r o t e i n (see S t u b b e a n d v a n d e r D o n k , 1998). D u r i n g the p a s t d e c a d e , b e g i n n i n g with t h e i d e n t i f i c a t i o n o f 2 , 4 , 5 - t r i h y d r o x y p h e n y l a l a n i n e ( T P Q ) , m a n y m o r e e x a m p l e s o f m o d i f i e d a r o m a t i c a m i n o acids have c o m e to light. T h e s e vary f r o m r e p l a c e m e n t o f a h y d r o g e n o n t h e tyrosine r i n g by a s e c o n d a m i n o a c i d side c h a i n (see t h e c h a p t e r by R o g e r s a n d D o o l e y in this v o l u m e ) to t h e h y d r o x y l a t i o n o f t r y o s i n e a n d tryptop h a n r e s i d u e s to p r o d u c e p r o t e i n - b o u n d q u i n o n e s . C u r r e n t l y , t h e r e are f o u r k n o w n q u i n o n e c o f a c t o r s t h a t use reactive c a r b o n y l s to catalyze a m i n e o r a l c o h o l o x i d a t i o n . T P Q (Janes et al., 1990) (Fig. 1A) f o u n d in c o p p e r a m i n e o x i d a s e s ( C A O s ) a n d lysine t y r o s y l q u i n o n e ( L T Q ) ( W a n g et al., 1996) (Fig. 1B) f o u n d in lysyl oxi-

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142

JOANNEE. DOVEANDJUDITH E KLINMAN

(A)

(B)

H

0

(C)

o

o2H

0 FIG. 1. Structures of the quinone cofactors. (A) 2,4,5-trihydroxyphenylalanine (TPQ); (B) lysine tyrosyl quinone (LTQ); (C) pyrroloquinoline quinone (PQQ); (D) tryptophan tryptophylquinone (TTQ).

dase (LOX) are discussed in this chapter. Both of these enzymes oxidize primary amines to the corresponding aldehyde, and then transfer the reducing equivalents to dioxygen to form hydrogen peroxide. From alignment of protein and cDNA sequences (Mu et al., 1992; Wang et al., 1996), it can be concluded that T P Q and LTQ are formed by a posttranslational insertion of oxygen into an active-site tyrosine. For TPO~ and likely LTQ, this is a self-processing reaction facilitated by an activesite copper ion and the ability to react with dioxygen. Pyrroloquinoline quinone (PQQ) (Fig. 1C) and tryptophan tryptophylquinone (TrQ) (Fig. 1D) are found in enzymes that catalyze the oxidation of alcohols and amines, respectively. Among the now established family of quino cofactors, P Q Q is the sole, low-molecular-weight, noncovalently attached structure, behaving as a "classical" cofactor, that is, not derived from a side chain of its cognate protein. A feature common to P Q Q and T r Q is that the reduced forms of these cofactors are reoxidized by long-range electron transfer to another protein rather than to molecular oxygen. It also appears that both P Q Q and TlrQ are formed by corn-

TPQ AND LTQ

143

plex biosynthetic pathways invoMng multiple gene products. These cotactors are the subject of the chapter by Davidson in this volume. TPQ, LTO~ and T T Q are examples of an e x c i f n g area of enzymoloD' in which new functionalities derived from the posttranslational modification of amino acid side chains expand the chemical capabilities of an enzyme beyond the natural amino acids./n situ formation of these vet} reactive cofactors may be necessary to ensure their stability and prevent unwanted side reactions. As a result, a molecule such as T P Q can be used effectively for redox chemistry, even though the free molecule is a known neurotoxin (Janes et al., 1990). This chapter focuses on recent advances in understanding the mechanisms of catalysis and T P Q formation in GAOs. For earlier work in the field, see reviews by Mclntire and Hartmann (1993); Klinman and Mu (1994); and Dooley (1999). We also discuss the progress in understanding the biological roles of these enzymes. Although the mechanisms of catalysis and cofactor formation in LOX are less well developed, the available data for LOX are considered in the context of the CAOs. Finally, the sequences and possible biological roles of a recently reported family of LOX-like enzymes are described.

II. TOPA QUINONE(TPQ) Although it had been known for decades that CAOs contain both copper and an organic cofactor, the structure of the organic cothctor proved elusive (Klinman and Mu, 1994). Initially, different known cofactors were considered candidates, including pyridoxal phosphate and PQQ; however, no cofactor was consistent with all the properties of the CAO cofactor (Knowles and Yadav, 1984). In 1990, biochemical and spectroscopic analysis of an isolated, active-site peptide that contained tile derivatized cofactor was used to elucidate the structure. The cofactor was demonstrated to be 2,4,5-trihydroxyphenylalanine quinone (topa quinone or TPQ) (Fig. 1A) (lanes et al., 1990).

III. COPPERAMINE OXIDASES A. Enzyme Structure CAOs are homodimers composed of approximately 70 to 90 kDa subunits (Klinman and Mu, 1994). The first solved crystal structure of a CAO was that of the 1"2. coli enzyme (ECAO) (Fig. 2A). This structure showed that the enzyme is primarily [~ sheet with an overall stnlcture

144

JOANNE E. DOVE AND JUDITH P. KLINMAN

A

%,

B

FIG. 2. Crystal structures of copper amine oxidases from (A) Escherichia coli (Parsons etal., 1995) and (B) Hansenulapolymorpha (Li etal., 1998). One subunit of the homodimer is shown in black and the other in gray. Pictures were generated using Insight II; the side chains are not displayed.

that looks like a m u s h r o o m with a cap a n d stalk (Parsons et al., 1995). To date, the crystal structures o f t h r e e o t h e r CAOs [pea seedling a m i n e oxidase (PSAO) ( K u m a r et al., 1996), Arthrobactor globiformis p h e n e t h y l a m i n e oxidase (AGPAO) (Wilce et al., 1997) a n d Hansenula polymorpha a m i n e oxidase (HPAO) (Li et al., 1998)] have b e e n c o m p l e t e d , revealing a very similar fold to ECAO in the cap, b u t lacking the stalk o f ECAO (see HPAO structure in Fig. 2B).

TPQAND LTQ

14~

Within the active site of each subunit, a mononuclear Cu(II) ion is liganded by three fully conserved histidine residues and by two waters in a square pyramidal geometry (Parsons et al., 1995). In the reactive form of the enzyme, TPQis near to, but not a ligand of, the Cu(II). Tile C5 carbonyl, which will react with substrate amine, is oriented toward the active site base (D319 in the H. polymorpha sequence1). In this orientation, T P Q has a characteristic absorbance centered around 480 n m similar to that seen in TPQ model compounds. In the resting enzyme, this reactive conformation has been seen unambiguously in the HPA() crystal structure (Fig. 3A) (Li et al., 1998). The relevance of this orie~ltation for catalysis is supported by its observation in an ECAO crystal structure with a b o u n d substrate mimic (Wihnot et al., 1997). Either a 180 ° flipped or a disordered cofactor orientation has been observed in the other crystal structures. These enzymes still have tire 480 mn absorbance of oxidized T P Q and are able to react with amine, although this is believed to require accessing the reactive conformation. In PSAO, the flipped orientation may result from the acidic pH used for enzyme crystallization and suggests a role fi)~" active site prototropic states in maintaining the reactive conformation of the cofactor (Kumar et al., 1996). In ECAO and AGPAO, disorder was seen in the resting state of the cofactor, which could be m o d e l e d as a combination of the reactive and flipped conformation (Parsons et al., 1995; Wilce et al., 1997). The different T P Q confoimations observed anaong HPAO, ECAO, and AGPAO may reflect subtle diiterences in the active site, although the structural cause ()f this p h e n o m e n o n is n o t known. In addition to these conformations in which TPQ is not bound to the metal, an inactive conformation has been observed ira ECAO and AGAO in which the TPQhas rotated and become a ligand to the copper (Parsons et aL, 1995; Wilce et al., 1997). This structural change can be induced by ammonium ion and results in a yellow absorbance for the enzyme, as opposed to the usual pink (ca. 480 nm) absorbance of oxidized TPQ. Intriguingly, the accessibility of this conformation has been suggested to reflect the need for the precursor tyrosine (Y405) to ligand the copper during cofactor formation (see Section III,C) (Wilce et al., 1997). Resonance Raman spectroscopy has been used as a sensitive probe for the structure of TPO~ as well as the characterization of intermediates d u r i n g the catalytic cycle. Spectra of underivatized T P Q showed that only one carbonyl (C5) is able to u n d e r g o isotopic labeling with

I All residue n u m b e r s r e p o r t e d are based on the HPAO sequence,

] 46

JOANNE E. DOVEANDJUDITH P. KLINMAN A

B

•.~ N~4~

E406

D319~,~pQ y305/H456~Cu(II) •

H624

C

TI'Q~5 N~04

p'"2.s0A

~DIN

FIG. 3. Hansenulapolymo~pha amine oxidase active-site structures from the (A) coppercontaining mature enzyme (Li et al., 1998) and (B) zinc-containing precursor protein (Chen et al,, 2000). (C) View of the hydrogen bonding network connecting E406 and N404 that reduces the mobility of TPQ during turnover. One subunit is in black and the other is in gray. All pictures were generated using Insight II.

TPQaN~LTQ

147

H ~ 8 0 (Mo~nne-Loccoz et al., 1995). This result, c o m b i n e d with model studies (Mure and Klinman, 1993), has implicated the C5 position as the site of nucleophilic attack by substrate during catalysis (see Section III,B, below). Isotopic substitution was observed at the C3 position when the enzyme was incubated in D20. The exchange supports a T P Q structure in which an anion is delocalized between the C2 and C4 carbonyls (Janes et al., 1990; Mo6nne-Loccoz et al., 1995). This delocalization probably favors the p-quinone, as X-ray crystal structures indicate a distance of ca. 2.5 A between the the oxygen at C4 of cofactor and the hydroxyl group of a conserved tyrosine in the active site (Tyr-305). B. Catalysis

CAOs catalyze the oxidative deamination of primary amines to the corresponding aldehyde concomitant with the reduction of molecular oxygen to hydrogen peroxide via the following reaction: E-TPO~,x + R-CH2-NH3 + -+ E-TPO~-~d + RCHO (Reductive Half-Reaction) E-TPQxed + Oz --+ E-TPO~,x + H202 + NH4+ (Oxidative Halt-Reaction) 1. Reductive Half-Reaction

The proposed mechanism of the reductive half-reaction, in which the amine is oxidized to the aldehyde, has been well established and is shown in Scheme 1. A channel through the protein, which allows amines to approach T P Q proximal to the C5 carbonyl and active site base, has been observed crystallographically in CAOs from AGPAO, PSAO, and HPAO (Kumar et al., 1996; Wilce et al., 1997; Li et al., 1998). In ECAO, the channel appears to be partially obstructed by a side chain (Wilce et al., 1997). The pH profile of hcat/K M (amine) suggests that the amine must be in the protonated state on initial encounter with the enzyme (Farnum et al., 1986). Interaction of the protonated substrate with the protein is facilitated by a negatively charged patch of amino acids found at the outside of the proposed substrate channel (Wilce et al., 1997). The amine must then be d e p r o t o n a t e d for nucleophilic attack at the C5 carbonyl of T P Q to form the carbinolamine (Scheme 1A). It seems likely that the active site base (D319 in HPAO), participates in amine deprotonation and subsequent transfer of this proton to the C5 oxygen of TPQ.

148

JOANNE E. DOVE ANDJUDITH

P.

KLINMAN

(A)

(B)

+

+,

OH

O

/

CU(II)

"Y" ~)

+ H "N--C--R H lu

-

(c)

%/

(O)

H÷

~ .o

+ / "5" -N=C--R / Cu(ll) ~)H H H .rrt - Asp

H20

o R--~H .o

+ (Cu(,I) ~ "NH, OH _ "rd ~ Asp

SCHEME 1. Proposed mechanism for the reductive half-reaction of CAOs.

Dehydration of the carbinolamine (Scheme 1B) results in the formation of the protonated substrate imine intermediate. The first evidence for this intermediate came from reductive trapping experiments with sodium cyanoborohydride (Hartmann and Klinman, 1987). Later, this intermediate was observed in BSAO using rapid-scanning stopped-flow, where the protonated, substrate imine of benzylamine was observed at 340 nm (Hartmann et al., 1993). Proton abstraction from the t~ carbon of the substrate imine (Scheme 1C) generates the product imine. An isotope effect o n k c a t / KM (amine) in enzymes from a n u m b e r of sources suggests that this step partially or fully limits the rate of the reductive half-reaction (Palcic and Klinman, 1983; Hevel et al., 1999). The primary isotope effect in BSAO is 12, a value that is larger than semiclassical limits (Palcic and Klinman, 1983). A study of this p h e n o m e n o n over a range of temperatures has suggested that quantum mechanical tunneling is important in the transfer of both protium and deuterium and is responsible for the unusually large isotope effects (Grant and Klinman, 1989). An aspartate acts as the base that abstracts the proton. This was demonstrated from a crystal structure of ECAO in which T P Q was reacted with a sub-

TeQANDtXQ (A)

/

H OH

N~-c--cH--R H I ('~T

-Asp/

149

(B)

/

H OH

N--C=C--R H H

T--Asp/

(C)

£)H

N-C=C--R H H)H

+ / H " T " "N=C--C--RH H2 OH

H--~Asp S

-ASp/

H20

S(:nEMF2. Proposed mechanism for isotopic exchange at the (;2 position of amine.

strate mimic. The resulting structure is analogous to the substrate imine intermediate, and the active-site aspartate is clearly positioned for proton abstraction (Wilmot et al., 1997). Although all CAOs catalyze this proton abstraction during the reducrive half-reaction, enzymes from different sources catalyze the proton abstraction with varying stereospecificities; C•Os have been found that are selective for pro-R, pro-S, or nonselective (Coleman et aL, 1991). As the active sites of CAOs are highly conserved, the structural basis for this observation is not yet known. The p r o d u c t imine, f o r m e d after a-proton abstraction, does not accumulate significantly during turnover, leading to an inability' to be reductively trapped u n d e r steady-state conditions (Hartmann and Klinman, 1987) or be observed spectroscopically during the pre-steady state with benzylamine (Hartmann et al., 1993). Although a recent study in BSAO has suggested a relatively slow rate of hydrolysis, the determination of rate constants was based on the assumption that T P Q and the substrate imine have the same absorbance (Bellelli et al., 2000) ; the conclusions n e e d to be reevaluated in light of the expected absorbance of 480 nm for T P Q and 340 nm for the protonated substrate imine. The existance of the product imine was initially interred from experiments using substrates that are able to tautomerize from the product Schiff base. During turnover with these substrates, it was possible to detect spectroscopically an off-pathway species (Hartmann et aL, 1993). The relevancy of the product imine as an intermediate was additionally indicated by isotopic exchange at the amine C2 position during turnover (Scheme 2). The proposed mechanism for exchange is formation of a product Schiff base complex that then undergoes [~-proton abstraction by the active-site base (Scheme 2A). Using ~tritiated substrate, tritium can be exchanged with water (Scheme 2B) leading to loss

150

JOANNE E. DOVE AND JUDITH R KL1NMAN

of the label in product (Scheme 2C) (Farnum et al., 1986). In a separate study, C2 exchange was followed by determining the level of incorporation from D 2 0 during the course of turnover. Isotopic exchange could only be detected in the aldehyde product, not the substrate. This is significant because it suggests that, at least in some CAOs, proton abstraction leading to product Schiff base from the substrate Schiff base is essentially irreversible (Coleman et al., 1991). Subsequently, resonance Raman spectroscopy allowed the direct observation of the product imine in a mutant of HPAO, as well as in BSAO and AGAO, using methylamine as a substrate (Cai et al., 1997; Nakamura et al., 1997). The lack of D 2 0 sensitive peaks in the resonance Raman demonstrated that, in all cases, the product imiue was deprotonated. As noted above, hydrolysis of the protonated, product Schiff base along the reaction path is expected to be a rapid step. The ability to observe the product Schiff base is attributed to the formation of "off-path" species that arise due either to mutation of the enzyme or the use of a nonoptimal substrate. It has been postulated that rotation of the relatively small product Schiff base formed from methylamine occurs concomitant with deprotonation, leading to accumulation of a species that cannot normally be detected under turnover. Hydrolysis of the protonated, product imine (Scheme 1D) generates aldehyde and aminoquinol, the substrate reduced form of TPQ. The absorbance of the aminoquinol has been shown to be 310 nm (Mure and Klinman, 1993). Model studies have also shown that the pKas of the C2 and C4 hydroxyl groups in the aminoquinol are 9.6 and 11.6 (Mure and Klinman, 1993). If these values are not significantly perturbed in the enzyme, both of these oxygens will be protonated on reduction, resulting in the storage of two electrons and two protons in the cofactor following amine oxidation.

2. Oxidative Half-Reaction The oxidative half-reaction involves the reoxidation of the aminoquinol by molecular oxygen to generate TPO~ ammonia, and hydrogen peroxide. The mechanism for this half-reaction has been controversial and much current work is being focused on this question. Two mechanistic proposals have been put forth that differ both with regard to the role of the copper and the nature of the 02 binding site. Both proposals are illustrated in Scheme 3.

a. Electron Transfer from Aminoquinol to Oxygen. Klinman and co-workers have suggested a mechanism in which molecular oxygen first binds to the enzyme in a nonmetal binding site, followed by electron transfer from the aminoquinol directly to molecular oxygen, and stabilization of

TPQANDvrQ

151

(B) NH2

NH2

(o) (F)

(G)

H202

.o

.o

L~ -c_u(H> OH NH2

O~

C~)

OH NH

C~(ll)

\ b~'-

H20

__Lo OH

I)

NH~.

_Xo O-

11

O"

5,

S(:HEME3. Proposed mechanismsfor the oxidativehalf-reactionof CAOs.

the resulting superoxide by Cu(II) (Scheme 3A, B, C) (Su and Kilnman, 1998; Mills and Klinman, 2000). This mechanistic proposal was originally based on IsO kinetic isotope effects in bovine serum amine oxidase (BSAO) (Su and Klinman, 1998), as well as HPAO (Su, 1999), which suggested that the first electron transfer into molecular oxygen was rate-limiting in the oxidative half-reaction. From the lack of a solvent isotope effect, electron transfer was concluded to be uncoupled from proton transfer. It was noted that the observed ~sO isotope effect could have been consistent with a bimolecular collision between oxygen and Cu(I) to form Cu(II) and superoxide (Scheme 3E). However, a rate-limiting bimolecular reaction would be expected to show a reduction of rate with increasing viscosiw of the solution, whereas /~-at//~l (O~) was shown to have a rate independent of added viscosogen. Additionally, a rate-limiting reaction between oxygen and Cn(I) would imply an accumulation of Cu(I) and the aminosemiquinone, as generation of these species would precede the slow step. It has been shown with BSAO that, in fact, aminoquinol absorbance decays in the rate-limiting step of the oxidative half-reaction (Su and Klinman, 1998). In a separate suldy of BSAO, the reaction of aminoquinol with dioxygen, monitored by stopped-flow, could only be fit by including a step prior to electron transfer (Bellelli et al., 2000). When taken together, all of the evidence points toward prebinding of molecular oxygen at a nonmetal site (Scheme 3A), followed by a rate-limiting electron transfer from the aminoquinol to tool-

152

JOANNE E. DOVE AND JUDITH P. KLINMAN

ecular oxygen (Scheme 3B), at which point superoxide undergoes stabilization by Cu(II) (Scheme 3C). Very recent studies, in which Cu(II) has been replaced by Co(II) in HPAO, lend further support to this proposed mechanism (Mills and Klinman, 2000). Values of kcat for the copper and cobalt-containing enzymes have been shown to be essentially identical; because a Co(I) state is not accessible, this rules out a mechanism in which the metal undergoes an obligatory +2 to +1 valence change. The properties of kcat/K M ( 0 2 ) for reaction of the cobalt-containing enzyme appear very similar to the copper enzyme, with regard to the size of the lsO isotope effect and the lack of a significant effect of solvent viscosogen on rate. However, k~at/KM (O2) has been shown to be approximately two orders of magnitude reduced from wild type. This has been suggested to result either from an alteration in the O2 binding site in the cobalt-containing enzyme, or from an elevation of the pKa for cobalt-bound H 2 0 (Mills and Klinman, 2000). Prebinding of oxygen prior to electron transfer requires a nonmetal oxygen binding site. This site has been proposed to be a hydrophobic pocket located near the Cu(II) in the HPAO crystal structure (Li et al., 1998). Mutation of a residue in this pocket, M634, does show a reduced kcat/KM ( 0 2 ) , with the cause of the rate reduction u n d e r investigation (Goto and Klinman, unpublished data). Although the mechanism in Scheme 3 (A, B, C) is unusual, precedence for a nonmetal binding site for dioxygen, as well as direct reaction between an organic cofactor and molecular oxygen, can be found in other enzyme systems (Klinman, 2001).

b. Electron Transfer from Reduced TPQ to Cu(II). Dooley and co-workers have suggested a different mechanism for cofactor reoxidation in which electron transfer from aminoquinol to Cu(II) generates aminosemiquinone and Cu(I). An electron can then be transferred from Cu(I) to molecular oxygen through an inner sphere mechanism to generate Cu(II) and superoxide (Scheme 3D, E) (Dooley et al., 1991). Indeed, following the addition of amine anaerobically, the aminosemiquinone has been show to accumulate to a significant extent in CAOs from a n u m b e r of sources (Dooley et al., 1991). A temperature j u m p EPR experiment was used to demonstrate that in PSAO and ArthrobactorP1 amine oxidase (APAO) the rate of electron transfer was kinetically c o m p e t e n t to be on the reaction pathway (Turowski et al., 1993; Dooley and Brown, 1996). The mechanism in Scheme 3 (D, E) further predicts that ligands that bind to copper should be compet-

TPQAND LTQ

f 53

itive with molecular oxygen. While this has been reported for pig plasma amine oxidase (Barker et al., 1979), recent studies with HPAO indicate noncompetitive behavior between azide and 02 (Schwartz et al., 2001). The ability of the aminosemiquinone to form in a thermodynamically favorable and rapid reaction suggested that it may be an intermediate in the oxidative half-reaction of these CAOs although accumulation, u n d e r noncatalytic conditions, leaves open the possibility that the species detected is off the reaction pathway. The rate of reoxidation of the substrate-reduced form of the CAO from lentil seedling (LSAO), a protein in which there is significant accumulation of the aminosemiquinone, was determined (Medda et al., 1998)However, the form of the enzyme that reacted with oxygen is not known. More conclusive support for this mechanism may come from a detailed kinetic analysis of catalysis. At this point, the mechanism of the initial electron transfer into dioxygen is still a matter of some debate, and further experiments will be necessary to demonstrate whether formation of Cu(I) is an obligatory step in the catalytic turnover of any CAO. Although the two mechanisms shown in Scheme 3 can be thought of as limiting electronic cases, there are substantial differences between them, in particular whether ligand reorganization terms that accompany a change in metal valence (present in D and E, but not A-C) contribute to the reaction energy barrier. The direct transfer of an electron from the aminoquinol to Oz has been shown to occur in solution in the absence of added metal ion (Mills and Klinman, 2000), and comparison to k,..~t/K~1 ( 0 ~ ) for HPAO indicates a ca. 4 order of magnitude rate acceleration, attributed to electrostatic catalysis by the active site copper in superoxide anion formation (Mills and Klinman, 2000). We note that while it seems most likely that a single mechanism will be followed in all C~Os, the possibility exists that CAOs from different sources generate superoxide and aminosemiquinone differently. Once superoxide has formed, reaction with the aminosemiquinone proceeds to generate iminoquinone and hydrogen peroxide. This requires a transfer of two protons and one electron from the aminosemiquinone to superoxide (Scheme 3F). The source of the two protons has been suggested to be the C2 and C4 oxygen of the mninoquinol (Li et al., 1998). Both of these oxygens will be deprotonated on formation of the oxidized cofactor due to pK~, changes, and a hydrogen bonding network from each oxygen to the copper site has been found in HPAO (Li et al., 1998).

154

JOANNE E. DOVE AND JUDITH P. KLINMAN

The deprotonated iminoquinone, with a 420 n m absorbance, was observed to accumulate in the HPAO active-site base mutant D319E (Plastino et al., 1998). This accumulation has been attributed to increased mobility and deprotonation of the iminoquinone, arising from an alteration in the ionic interaction that normally occurs between the positively charged, protonated iminoquinone with D319. During productive turnover, the protonated iminoquinone is expected to undergo rapid hydrolysis. Recently, iminoquinone has been directly observed in ECAO crystals that were freeze trapped following aerobic incubation of the enzyme in the presence of excess amine. The structure captures an exciting snapshot of the protonated iminoquinone, as well as peroxide liganded to copper prior to its protonation and release from enzyme (Wilmot et al., 1999). The iminoquinone can then be hydrolyzed to TPQ (Scheme 3G). It is also possible that an attack of a new amine substrate directly generates the substrate imine during turnover. There are currently no data to suggest whether hydrolysis or a direct transimination occurs, although the latter has been shown to be operative in the TTQ-containing methylamine dehydrogenase (Zhu and Davidson, 1999). C. TPQ Biogenesis 1. Posttranslational Oxidation of Tyrosine The mechanism for the creation of the novel cofactor, TPQ, has been an intriguing question since its discovery. The sequence of the activesite peptide from BSAO initially used to identify the cofactor is Leu-AsnX-Asp-Tyr, where X is the position of T P Q (Janes et al., 1990). Comparison of this amino acid sequence with the cDNA sequence revealed that a tyrosine was coded for at the position where TPQ was found in the protein (Mu et al., 1992), suggesting that TPQwas formed by the posttranslational oxidation of a specific tyrosine in the active site. Two mechanisms were initially proposed for the modification of the tyrosine. Either an enzyme with tyrosine hydroxylase-like or tyrosinase-like activity could catalyze the oxidation, or the oxidation could occur in an autocatalytic process (Mu et al., 1992). An early piece of evidence to address this mechanistic question came from the heterologous expression of active HPAO in S. cerevisiae. This yeast does not contain an endogenous CAO and, therefore, would not be expected to contain any additional enzymes required for TPQ formation (Cai and Klinman, 1994a). Thus, this argued that TPQ formation occurred in an autocatalytic process. Another insightful study involved the site-directed mutagenesis of a copper ligand, H456D, which prevented copper binding and TPQ formation

TPQ AND LTQ

155

(Cai and Klinman, 1994b). Copper would not be expected to be essential for ~rosine modification by an additional e n ~ m e , but would very likely be necessary for a self-processing reaction. The point was conclusively answered by Tanizawa and co-workers who isolated copper-free enzyme from a heterologous expression system and f o u n d that the precursor tyrosine was unmodified. TPQ could then be formed in vitro by the addition of copper, in the presence of molecular oxygen, to the purified apoenzyme (Matsuzaki et al., 1994). 2. Stoichiometrv

Knowledge of the overall stoichiometry of the biogenesis process is an important first step in establishing a mechanism. Dooley and coworkers have shown that for AGPAO the reaction consumes two moles of oxygen and produces one mole of hydrogen peroxide, per TPQ formed (Ruggiero and Dooley, 1999). This has two important implications. First, as tyrosine is oxidized by six electrons to form TPO~ the stoichiometry f o u n d with AGPAO results in a balanced equation and rules out the need for any external reductants. Second, as suggested by DooIcy and co-workers, the stoichiometry of the reaction provides a framework for envisioning the molecular mechanism for this transformation (Ruggiero and Dooley, 1999). For instance, previous proposals had included dopa as an intermediate, but that mechanism would predict the tbrmation of two moles of hydrogen peroxide. 3. Proposed Mechanism

A current proposal for the biogenesis mechanism is shown in Scheme 4. To separate copper binding from the subsequent reaction of enzyme with oxygen, it has proven useful to prebind copper anaerobically. It was initially envisioned that tyrosine would be activated for reaction with oxygen anaerobically, either through electron transfer to Cu(II) to form tyrosyl radical, or through liganding of tyrosine to Cu(lI). EPR and CD measuremenLs have revealed that greater than 90% of the copper is in the +2 oxidation state (Ruggiero et al., 1997). Mso, the anaerobic generation of nitric oxide in the presence of copper-bound precursor protein is fbund to not affect the final rate of TPQ production (Dove et al., 2000), suggesting that little or no organic radical is accumulating under these conditions. Both observations are inconsistent with the presence of a significant amount of Cu (I) or tyrosyl radical. In the event of a tyrosinate liganded to Cu(lI), a characteristic visible absorbance is expected; however, no stable visible absorbances are detected with copper p r e b o u n d to enzyme under anaerobic conditions (Dove et al., 2000), ruling out the accumulation of a tyrosinate-Cu(II)

156

JOANNE E. DOVE AND JUDITH P. KLINMAN

(A)

(c)

(B)

H30

OH , . ~

(D)

H20 His-cul (ll) His" His

(E)

(F)

I His-Cu 10/O HIS" His

(G)

OH His-CuP(11} His' "His

(H)

0) O~

-

L

OC H20 ~ His_cul(ll) His" His

0 0

+

HO OH OH2 His_Cul(!I)_OH2 OH His" His

H202,H*

k

OH2 0 His_C/(II}_OH2 OHis" "His

SCHEME 4. Proposed m e c h a n i s m for T P Q biogenesis.

species as well. The only species that could be detected spectroscopically u n d e r anaerobic conditions is a presumed Cu(II) binding intermediate, which has transient absorbance at 380 nm, and does not represent an intermediate in tyrosine oxidation (Dove et al., 2000). Taken as a whole, there is no evidence for an activated tyrosine species in the absence of 02 (Scheme 4A). Although copper added to the precursor protein in the absence of O 2 remains in the +2 oxidation state, the metal does appear to have a different geometry in the precursor protein than in the mature protein. Differences in the EPR hyperfine splitting of the Cu(II) signal (Ruggiero et al., 1997), as well as a blue shift of the Cu(II) d-d transition in the CD spectrum (Ruggiero et al., 1997), suggest a more tetrahedral environment for Cu(II) in the precursor protein. This is in contrast to the square pyramidal geometry observed in the TPQ-containing

TPQ AND IXQ

157

enzyme. This change in Cu(II) geometry may explain the difference in /~ for azide binding observed between precursor and mature HPAO (Schwartz et al., 2001). Recent kinetic investigations of biogenesis in HPAO have been undertaken in order to learn more about the interaction of oxygen with the enzyme. Using an assay protocol where an aerobic solution of precursor end,me is mixed with copper ion to initiate reaction, the rate of biogenesis was shown to be independent of solvent viscosity, indicating that 02 interacts with enzyme prior to the rate-determining step in biogenesis (Scheme 4B) (Schwartz et al., 2000). This is in contrast to an earlier proposal of a simple bimolecular reaction between molecular oxygen and the enzyme (Ruggiero et al., 1997), and is similar to the proposal for the oxidative half-reaction of catalysis (Scheme 3A). There are several lines of evidence indicating that a binding of Oz to protein promotes liganding of the precursor tyrosine to Cu(II) (Scheme 4C), This mechanistic picture of biogenesis emerges from crystallographic, spectroscopic, and kinetic analyses. The first apo-CAO crystal structure to be solved was apo-AGAO (Wilce et al., 1997). This structure is nearly superimposable with the mature en'zyme except that the 0 4 ox> gen of the precursor tyrosine was found to be pointing toward the vacant copper site rather than toward Y305 (See Fig. 3A). The position of tyrosine was noted to be very similar to the position of T P Q in the inactive tbrrn of the holoenzyme (see Section III,A). The authors suggested that an activation of tyrosine by liganding to copper was likely to occur during biogenesis (Wilce et al., 1997). Very recently, the precursor HPAO structure was solved in which Zn(II) occupies the normal Cu(lI) site, and the precursor tyrosine remains unmodified (Chen et al., 2000). In this structure, the precursor tyrosine can be seen to ligand the Zn(II) (Fig. 3B). This results in a tetrahedral geometry, where the remaining three ligands are the histidines that ligand Cu(II) in the holoenzgame. This structure is believed to represent a snapshot of the precursor protein just prior to biogenesis. Besides the precursor tyrosine, M634 has rotated toward L425, precluding the binding of the two water molecules seen in this location in the holoenzyme. The significance of this motion is not certain, but it is interesting to note that M624 and L425 have been suggested to be the site of 02 binding during the oxidative half-reaction. At the same time, two new waters appear in the substrate channel at positions occupied by TPQ in the mature enzyme. These waters have been postulated to represem a site for 02 binding during biogenesis (Chen et al., 2000). Further evidence supporting a tyrosinate-Cu(II) intermediate comes from detailed spectroscopic and kinetic studies of biogenesis in HPAO. A small, but reproducible, 350 nm absorbance is observed prior to the

158

JOANNE E. DOVE AND JUDITH P. KLINMAN

0.024 0.02 0.016 oo 0.012 < "~ 0.008

..Q

0.004

0

o -0.004

,

300

,

400

500

600

700

800

Wavelength (nm) FIG. 4. Absorbance changes during TPQ biogenesis. Spectra were generated by prebinding Cu(II) anaerobically to apo-HPAO. Biogenesis was then initiated by exposure to air, Spectra shown are at (a) ! min and (b) 16 min following exposure to 02. An intermediate is observed (350 nm) prior to the formation of TPQ (480 nm). The sample was 40 btM enzyme in 50 mM HEPES, pH 7.0 at 25°C.

formation of T P Q (see Fig. 4). This intermediate is formed only in the presence of oxygen (Dove et al., 2000) and more rapidly than the observed rate of oxygen consumption. A similar intermediate with a 40 nm red-shifted wavelength has been observed in a copper ligand mutant (H624C) (Dove et al., 2000). Both the change in ~,m~x and the reduced rate of breakdown of the intermediate in the H624C mutant support direct involvement of copper in this species. Thus, this intermediate has been postulated to be a tyrosinate-Cu(II) species (Dove et al., 2000; Schwartz et al., 2000). The charge transfer complex between Cu(II) and tyrosinate is expected to contain some tyrosyl radical and Cu(I) character arising from resonance (Scheme 4D). This partial radical character is postulated to be essential for reaction with molecular oxygen and to control the rate of chemical reaction of the presursor protein with O2 (Scheme 4E), which is relatively slow in relation to catalytic turnover. The contribution of the extreme resonance form represented as tyrosyl radical will be diminished by replacement of H624 by the electron releasing cysteine, consistent with the decreased rate of breakdown of the charge

TPQAYDLTQ

159

transfer complex in the HPAO mutant H624C. Overall, it appears that the electrophilicity of the metal exerts a major influence on the rate of T P Q biogenesis (Dove et al., 2000). It is not possible to distinguish whether molecular oxygen will react first at the copper site or the tyrosinate/tyrosyl radical. In either case, the reaction is expected to lead to a metal-complexed peroxy intermediate (Scheme 4E) that can break down to give dopa quinone and hydroxide ion (Scheme 4F). Thus far, there is no experimental evidence to support these intermediates, but future etforts will undoubtedly be directed at detecting these species. Following the formation of dopa quinone, the conversion to T P Q involves the addition of hydroxide ion; this reaction occurs in model systems (Mure and Klinman, 1993). The reaction proceeds more rapidly above p H 8.5 and, therefore, has been suggested to involve the nucleophilic attack of hydroxide, rather than water, to give topa (Scheme 4H). In the enzyme, metal complexation of water is expected to lower the P/£a and promote this reaction. The proposed attack of hydroxide on dopa quinone to generate the C2 oxygen of T P Q is consistent with resonance Raman experiments in which the biogenesis reaction was conducted in H2~80 and the C2 oxygen of T P Q w a s found to be isotopically labeled (Nakamura et al., 1996). The oxidation of topa to T P Q by molecular oxygen (Scheme 41) is analogous to the oxidative half-reaction of catalysis, and is expected to proceed via a similar mechanism. D. Roles of Conserved Active Site Residues in Catalysis and Biogenesis CAOs must accomplish the dual functions of oxygen insertion into tyrosine (monooxygenase activity), as well as the oxidation of both topa during biogenesis and aminoquinol during turnover (oxidase activity'). Characterization of the effects of mutations of active-site residues has begun to allow us to address the role of the enzyme in facilitating these different processes. Table I summarizes the relative effects of different mutations of HPAO on turnover, the reductive half-reaction, the oxidative half-reaction, and biogenesis. 1. Catalysis T P Q must be in the active conformation for efficient catalysis, and many HPAO residues have been shown to be involved in positioning T P Q (see Fig. 3A). Mutants at N404, the residue immediately prior to TPQ, and D319, the active-site base, both have a substantially reduced k,.~t/K~a that was attributed to increased T P Q mobility (Plastino et al.,

160

JOANNE E. DOVE AND JUDITH P. KLINMAN

TABLE I Effect of Active-Site M u t a t i o n s in H P A 0 on Catalysis a n d Biogenesis ~

Mutation N404A e N404D

~at

hcat/ KM (Am)

kcat/ KM(O2)

0.09

9 × 10-4 ND

ND

Mobility b

kbio

0.02

TPQ, PI, AQ

ND ~

ND

ND

0.008 ND

Reference Schwartz et al., 1998 Dove et al., 2000

E406N ~

0.2

0.5

ND

PI

E406Q e

0.2

0.2

ND

PI

0.2

Cai et aL, 1997 Dove, 2000; Dove et al., 2000

D319E e

0.005

0.01

ND

TPQ, I Q

0.01

Plastino et aL, 1999

D319N

NA d

NA

NA

TPQ

ND

Plastino et al., 1999

D319A

NA

NA

NA

ND

ND

Plastino, 1997

Y305A/-

0.4

0.3

0.2

PI

0.001

Hevel et al., 1999

Y305C t

0.2

0.1

ND

P1

ND

Hevel et aL, 1999

Y305Ff

0.008

0.002

ND

PI

ND

Hevel et aL, 1999

H624C

ND

ND

ND

ND

0.01

Dove et al., 2000

a Activities are expressed as a fraction o f wild type activity. With methylamine, kcat is 3.2 s-l, kcat/KM (amine) is 6.4 x 104 M-is -1, a n d kcat/KM (02) is 2.1 X 105 M-is ~1 (Schwartz et aL, 1998); with ethylamine, k~at is 20 s-1, kcat/KM (amine) is 5.2 x 104 M-is -1, a n d kcat/KM (02) is 6.6 x 105 M-is -~ (Hevel et al., 1999); the rate of biogenesis, koio is 0.08 m i n -1 (Dove et aL, 2000). b This c o l u m n indicates mobility in T P Q or any intermediate d u r i n g the catalytic cycle. PI is the p r o d u c t imine, A Q is the aminoquinol, a n d I Q is the i m i n o q u i n o n e . c ND, determined. a NA, n o activity. e Catalytic parameters were d e t e r m i n e d with m e t h y l a m i n e as the substrate. fCatalytic parameters were d e t e r m i n e d with ethylamine as the substrate.

1998; Schwartz et al., 1998). Even copper appears to play an important role in maintaining the correct orientation of the cofactor, as T P Q is unable to react with a substrate mimic in copper-depleted enzyme. However, the divalent metals Zn 2+, Co 2+, and Ni 2+ are able to facilitate this reaction, suggesting that the role of Cu(II) is strictly structural in this case (Mills and Klinman, 2000). Aside from the active-site base (D319), which has a mechanistic role in the reductive half-reaction, many other active-site residues maintain the proper positioning of the T P Q ring during the catalytic cycle. Mutation of N404 or E406, the residues before and after TPQ, respectively, as well as Y305, the residue in close hydrogen b o n d i n g distance with the 04 of TPO~ leads to mobility of the product imine with methylamine as a substrate and results in accumulation of an inhibitory species. This

XpQAYDLTQ

161

can be prevented by using larger substrates, such as ethylamine (Cai el al., 1997; Hevel et al., 1999; Schwartz et al., 1998; Dove, 2000), suggesting that a structural motion is necessary for the inactivation. The effect of changing Y305 to phenylalanine is much more dramatic than to either alanine or cysteine. The reason for this is believed to be a binding of water in the alanine or cysteine mutants that preserves a hydrogen b o n d to the 0 4 oxygen of cofactor, whereas this is precluded in the phenylalanine m u t a n t (Hevel et al., 1999). Less is known about the effect of active site mutations on the oxidative half-reaction. Mutation of N404 results in a reduced/~t/KM (02) because of an increase in mobility of the aminoquinol (Schwartz et al., 1998). With the mutant Y305A, k~at/KM (02) is much reduced with benzylamine as a substrate; however this has been attributed to a slow release of benzaldehyde, rather than a direct effect on the chemist~' of the oxidative halt: reaction (Hevel et al., 1999). At this point, only mutation of the active site base (D319E) has been shown to have an effect on the chemist~" thal leads to regeneration of TPQ, notably an increase in mobility of the iminoquinone that is detected as a reduced ~-~t (Plastino et al., 1998). Perhaps not surprisingly, the active-site base (D319) appears to be the most important residue for catalysis. Mutation of this residue to glutamate results in the largest reduction of k~t, as well as a significant reduction in the rate of the reductive half-reaction. More dramatically, mutations of. this residue to either asparagine or alanine are completely inactive (Plastino, 1997; Plastino et al., 1998). Of residues that have been mutated to date, only D319 appears to be absolutely essential for catalysis. Overall, the major effect of mutation of HPAO active-site residues on catalysis is an increase in mobility of either TPQ or intermediates in the catalytic cycle. This suggests that much of the active site is dedicated to maintaining the proper structure for efficient catalysis. We have, in fact, identified a network of active-site residues that is structurally orthogonal to the region of the active site containing the active-site base and copper ion (Fig. 3C). This network involves side chains from both subunits and is postulated to generate a "wall" behind the TPQ, ensuring the requisite rigidity for cofactor during catalytic turnover (Schwartz et al., 1998). Ring mobility may result from the need for flexibility at this position during biogenesis, and the recruitment of many residues to maintain proper ring orientation during catalysis is one way in which CAOs achieve a balance between the structural requirements of biogenesis and catalysis. 2. Biogenesis In biogenesis, the role of the copper ligands appears straightforward; these residues bind the metal, and influence its electronic interaction

162

JOANNE E. DOVE AND JUDITH P. KLINMAN

with the precursor tyrosine (see Section III,C) (Dove et al., 2000). Mutations of several other residues have been studied (see Fig. 3B). The amino acids preceding and following the precursor tyrosine form an NY-D/E consensus sequence in all CAOs. Both positions have been mutated, E406Q and N404D, resulting in significantly decreased rates of biogenesis (ca. one and two orders of magnitude, respectively). However, in contrast to WT, no intermediates accumulate during the course of biogenesis, suggesting a role for E406 and N404 residues in formation of the tyrosine to Cu(II) charge transfer complex (Dove et al., 2000; Schwartz et al., 2000). D319, the active-site base during catalysis, has also been mutated to investigate its role in biogenesis. D319E forms T P Q at a rate about 90fold reduced from WT, b u t the cause of this reduction in rate is not known (Plastino et al., 1998). Surprisingly, although D319N and D319A are inactive catalytically, they both contain T P Q (Plastino, 1997; Plastino et al., 1998). Although rates of T P Q formation have not been determined for the asparagine and alanine mutants, it is clear that D319 is not essential for biogenesis. Y305 is an active-site residue that makes a close hydrogen bond with the 0 4 oxygen of TPQ. The mutation of Y305 to alanine results in a three order of magnitude decrease in the rate of biogenesis (Hevel et al., 1999). Of the mutations studied to date, this is the largest effect. Although the source of the rate reduction is not known, the crystal structure of zinc-containing HPAO reveals that Y305 is in position to aid in the C-H bond cleavage necessary to generate dopa quinone from the peroxy intermediate (Scheme 4F). More studies will be necessary to test this possibility. Although characterization of effects of active site mutations on biogenesis is at an early stage, it is interesting to note that only mutations that prevent copper binding (Cai and Klinman, 1994b; Rob et al., 1995) or mutations to the precursor tyrosine itself (Choi et al., 1996) are completely incompatible with T P Q formation. At least with the investigations that have been p e r f o r m e d to date, active-site amino acid side chains have been found to enhance the rate of reaction, but do not appear to be essential. The only requirement for facilitating this reaction may be a protein structural scaffold that allows a precise interaction of tyrosine and copper in the presence of oxygen. E. Biological Roles

Discerning the multiple biological roles of CAOs, particularly in plants and mammals, is an exciting area in which considerable progress has been made.

TPQ AND LTQ

[ 63

1. Bacteria and Yeast

In single-celled organisms, the primary role for CAOs is the release of nitrogen and carbon from amines u n d e r nutrient-limiting conditions. A n u m b e r of CAOs were originally identified by growth of bacteria and yeast with an amine as the sole nitrogen source. This work has been reviewed previously (Mclntire and Hartmann, 1993). 2. Plants

Plant CAOs have been suggested to be involved in cross-linking structural proteins for cell wall formation during development and wound healing. The role of the CAO appears to be the generation of hydrogen peroxide (by the oxidation of putrescine), which can then be used as a substrate for the peroxidase that catalyzes the cross-linking. This proposal is based on the coexpression of CAO and peroxidase during development, as well as reduced cell wall cross-linking in the presence of CAO specific inhibitors (Rea et al., 1998). CAOs have also been suggested to play a role in programmed cell death (PCD), based on the observation of expression of CAOs in cells undergoing PCD (Moller and McPherson, 1998). More evidence is needed to demonstrate a clear connection, but this dual role for CAOs could provide the link between developmental PCD and secondary cell wall formation, two processes that are temporally coupled in plants (Moiler and McPherson, 1998). .L M a m m a l s

The in vivo substrates of mammalian diamine oxidases include histamine, putrescine, and polyamines. Histamine is catabalized by cell-sur[hce-associated diamine oxidases. As the release of histamine is involved in vasodilation and smooth muscle contraction, diamine oxidases are likely to play a role in controlling these processes. Extrace[lular diamine oxidases may also oxidize putrescine. The peroxide product of this oxidation has been suggested to be a signal for apoptosis during embryonic development. Intracellularly, putrescine promotes growth; oxidation by diamine oxidases inhibits this process (see Houen, 1999, and references therein) The mammalian membrane-bound monoamine oxidases were previously characterized as the semicarbazide-sensitive amine oxidases, and it has only recently been demonstrated that they contain T P Q as a cofactor (Holt et al., 1998). The in vivo substrate of these enzymes, as well as BSAO, is still a matter of speculation, but may include methvlamine, aminoacetone, tyramine, and dopamine, among others (IMes,

164

JOANNE E. DOVE AND JUDITH P. KLINMAN

1996). Unexpectedly, the cloning and sequencing of a vascular adhesion protein from humans revealed that it was the same as the membrane-bound m o n o a m i n e oxidase (Smith et al., 1998). It has now been shown that the m e m b r a n e - b o u n d monoamine oxidase in mouse also functions as a vascular adhesion protein (Bono et al., 1998). In this role, the glycosylation of the CAO has been shown to be essential for VAPmediated lymphocyte recruitment to endothelial cells (Salmi andJalkanen, 1996). It has not yet been shown, however, whether enzymatic activity plays a role in this process. It will be very interesting to see if this is a separate function for the CAOs in endothelial cells, or whether catalysis plays a role in the inflammation process. Studies of mature adipocytes indicate an increased population of CAOs on their outer cell surface, relative to the adipocyte precursor (Moldes et al., 1999). It has also been shown that hydrogen peroxide, one of the products of the CAO reaction, leads to increased movement of the glucose transporter to the membrane of adipocytes. This recruitment may occur because of increased intracellular protein phosphorylation resulting from an oxidative inactivation of tyrosine phosphatase by H202 (Czech et al., 1974; Enrique-Taranc6n et al., 1998). Ongoing studies in this laboratory indicate that turnover of aliphatic amines by the cell surface CAO of mature adipocytes leads to increased phosphorylation of intracellular proteins (Wertz and Klinman, 2000). In a diabetic condition with decreased uptake of glucose into cells, muscle and fat tissue begin to break down proteins as a source of energy. It is possible that the aliphatic amines, produced from this protein breakdown, are turned over by surface CAOs, resulting in the production of H202 and a biochemical result that is analogous to that of insulin.

IV. LYSlNETVROSVLQUINONE(LTQ) Although lysyl oxidase (LOX) contains copper as a cofactor and catalyzes a similar reaction to CAOs, a number of differences between the two enzymes suggested that the carbonyl cofactor in LOX might not be TPQ. LOX was much smaller than the CAOs (Klinman and Mu, 1994) and did not contain the conserved consensus sequence found in all CAOs (lanes et al., 1992). In 1996, biochemical and spectroscopic characterization of the isolated active-site peptide from bovine aorta LOX revealed the novel quinone cofactor, lysine tyrosylquinone or LTQ (Fig. 1B) (Wang et al., 1996). This cofactor is formed by the post translational oxidation of a tyrosine that is covalently linked to the E-amino group o f a lysyl side chain. Site-directed mutagenesis in C H O cells confirmed the requirement of the

TPQ AND LTQ

165

precursor tyrosine and lysine for enzyme activity (Wang et al., 1996). Finally, comparison of the resonance Raman spectra of underivatized LOX and an LTQ model compound provided unambiguous evidence for the assigmnent of the LTQ structure (Wang et al., 1997).

V. LYSYL OXIDASE

A. Enzyme Structure

LOX is a 32 kDa monomeric enzyme that has been purified from mammalian sources including bovine aorta (Kagan et al., 1979; Kagan and Sullivan, 1982; Williams and Kagan, 1985) and porcine skin (Shackleton and Hulmes, 1990; Cronshaw et al., 1993). Detailed characterization of the enzyme has been slowed by the fact that it is only soluble in the presence of urea and has been difficult to obtain in high yields (Kagan et al., 1979). Additionally, heterologous expression in C H O cells results in a yield that is too low for detailed kinetic and spectroscopic characterization of the enzyme (Kagan et al., 1995). There have been initial reports of expression of LOX in E. coli, but no further results have been reported in any of these systems (Ouzzine et al., 1996; Di Donato et al., 1997). At the moment, there is no X-ray crystal structure of the enzyme, and there has been no characterization of the effect of single point mutations on biogenesis or catalysis beyond the demonstration of the requirement of the precursor tyrosine and lysine for LTQ formation. The availability of a high-yield expression system would greatly accelerate progress in studying LOX and LTQ. LOX has major peaks in its resonance Raman spectra at 1683 cm -I, 1529 cm -l, and 1386 cm q , which are very similar in frequency to those seen in the LTQ model c o m p o u n d (Wang et al., 1997). Incubation of both LOX and the LTQ model c o m p o u n d in H21SO results in a shift in the ca. 1683 cm q peak to ca. 1656 cm -l. The size of the shift suggests that, as in TPO~ only one carbonyl is exchangeable with H 2 0 (Wang el al., 1997). This is supported by observation of only a two mass unit change following incubation of the model c o m p o u n d in H2180. Further, reaction of the model c o m p o u n d with the substrate mimic, phenylhydrazine, indicates that the C5 carbonyl of LTQ is susceptible to nucleophilic attack (Wang, S. X., Mure, M., and Klinman, J. p., unpublished data). Thus, amines are expected to react with the C5 carbonyl during catalysis. In the LTQ model c o m p o u n d , it has also been shown that the largest effect of incubation in D90 is on the ca. 1529 cm q peak, with a much smaller effect on the ca. 1386 cm -I peak (Wang et al., 1997). The assign-

166

JOANNE E. DOVE AND JUDITH E KLINMAN

ment of these D20 sensitive peaks to the C2 N H of the cofactor (Wang et al., 1997), in addition to a pKa analysis of model c o m p o u n d s (Wang, S. X., Mure, M., and Klinman, J. P., unpublished data), supports the assignment of LTQ as a neutral orthoquinone. This is in contrast to T P Q where the C2 oxygen has partial negative character. In the LTQ model c o m p o u n d , unlike TPQ, there is no evidence for C3 hydrogen exchange (Wang et al., 1997). The mechanism for this hydrogen exchange requires delocalization of the negative charge between the C2 and C4 oxygens of TPQ. Thus, an absence of exchange is also consistent with a neutral orthoquinone cofactor structure. Based on their very similar resonance Raman spectra, observations in the LTQ model c o m p o u n d are expected to hold true for LTQ within the enzyme (Wang et al., 1997). The presence of a carbonyl, rather than an anion, adjacent to the reactive C5 carbonyl is likely to be a significant mechanistic difference between the reactivities of LTQ and TPQ. O n e of the most striking features of CAOs is the mobility of T P Q during catalysis. LTO~ on the other hand, is not expected to be mobile because of the covalent cross-linking of the ring to a lysyl side chain. Initial experiments with the removal of copper from LOX seem to confirm this difference between the two cofactors. Following the removal of copper, LOX is still able to react with the substrate mimic, phenylhydrazine (Tang, C., and Klinman, J. P.). This is in contrast to CAOs, where T P Q moves into an unreactive conformation in the absence of copper (Mills and Klinman, 2000). B. Catalysis

Unlike CAOs, much is known about the biological role of LOX. The in vivo substrates of LOX are tropocollagen and tropoelastin. LOX oxidizes the ~-amino group of lysyl side chains forming c~-aminoadipic-8semialdehyde with concomitant reduction of dioxygen to hydrogen peroxide. Subsequent nonenzymatic condensation with other aldehyde or lysyl groups forms the covalent cross-links necessary for the biosynthesis of mature collagen or elastin (Smith-Mungo and Kagan, 1998). The proposed mechanism for catalysis is shown in Scheme 5. The binding site necessary for LOX to oxidize protein lysyl side chains is likely to be very different from CAOs, which oxidize small molecule amines. LTQ is expected to be more surface accessible than TPQ, at least prior to the binding of substrate. Two pK~s are observed on k~at/KM (amine). One has been assigned to the amine substrate with faster turnover observed under more acidic conditions (Gachern et al., 1988). As deprotonation of amines would be expected to facilitate nucleophilic

meQAYDIXQ

(A) ~ ~

167

(B)

RCH2NH3 f H

H20

0

His~

(c)

(D)

(E)

H20 R-CH •

.

\ A

His~

Hi$~

HiS~

SCHISMS"5. Proposed catalyticmechanismfor LOX.

attack to form the carbinolamine, the unexpected finding of faster catalysis with protonated amines suggests that amine protonation is important for either substrate binding or carbinolamine formation. Following the binding of substrate, the mechanism of LOX is likely to be very similar to CAOs. Nucleophilic attack of amine at the C5 carbonyl generates a carbinolamine (Scheme 5A). Subsequent dehydration results in a substrate imine (Scheme 5B). Unlike TPO~ there is no negative charge on the C4 oxygen of LTQ, which in the case of TPQ favors the maintenance of protonated substrate imine complex. Studies by Kagan and co-workers further suggest that the active-site base may be a neutral histidine (see below), rather than the anionic aspartate found in all of the copper amine oxidases. The maintenance of the protonation states of the substrate and product imines during catalysis may d e p e n d on negative charge stabilization provided by other residues within the active site. Proton abstraction fi-om the 0~-carbon of the substrate imine generates the p r o d u c t imine (Scheme 5C). With tyramine as a substrate, deuterium isotope effects of 2,6 o n kca t and 4.8 o n h , m / K M (amine) have been reported (Shah et al., 1993). These kinetic isotope effects suggest that proton abstraction partially or fully limits the reductive

168

JOANNE E. DOVE AND JUDITH P. KLINMAN

half-reaction, and that the reductive half-reaction partially limits turnover. Kagan and co-workers have suggested that a second pKa observed on ]~cat/KM (amine) (see above), which is independent of the pKa of the substrate, represents the ionization of an active-site base (Gacheru et al., 1988). The proposal of general base catalysis is supported by the observation of pro-S stereospecificity in the proton abstraction (Shah et al., 1993). The observed Pga of 7.0, combined with the enthalpy of ionization, led to the conclusion that the residue is a histidine (Gacheru et al., 1988). Additionally, reaction with diethylpyrocarbonate, a reagent that selectively reacts with histidines, caused inactivation of lysyl oxidase, and prior incubation with n-hexylamine protected against inactivation (Gacheru et al., 1988). Although all of this evidence is consistent with histidine functioning as the active-site base, either mutagenesis studies or an X-ray crystal structure are needed for more conclusive support. As with CAts, observation of C2 exchange (Scheme 2) supports the proposal of the product inaine as an intermediate (Shah et al., 1993). Hydrolysis of the product imine generates the aminoquinol and aldehyde (Scheme 5D). The cofactor is reoxidized by molecular oxygen to generate hydrogen peroxide and iminoquinone (Scheme 5E). Although the mechanism for this part of the reaction has not been investigated, it is likely to be very similar to the oxidative half-reaction of CAts (Scheme 3). As discussed for TPO~ substrate amine may either react directly with the iminoquinone form of LTQ or the iminoquinone may first undergo hydrolysis to LTQ and NH4 +. C. L T Q Biogenesis

During identification of the cofactor in LOX, Edman sequencing of the active-site peptide revealed that, in fact, two amino acids were present at each round of sequencing with one unidentifiable amino acid within each of the peptides. Comparison with the cDNA-derived sequence revealed that one position corresponded with a tyrosine and the other to a lysine, and suggested that LTQ was formed by the posttranslational modification of those active-site residues (Wang et al., 1996). LTQ is likely to be formed by a self-processing reaction based on a number of reports of expression of active LOX in E. coli cells (Ouzzine et al., 1996; Di Donato et al., 1997), which would not be expected to contain any processing enzymes. Like TPQ, LTQ may be formed in a copper-assisted process. By analogy to what is known about TPQ, a mechanism can be postulated for LTQ biogenesis (Scheme 6). Tyrosine

T P Q AND

LTQ

169

(c)

(B)

(A)

Cu0b

02

L.

o.

o.~

Q~ (s)

(F)

N~j~

Q~II) (3

S

/0 C~II)/0

@,

ll)

(D)

Q~ll)

OH

02 H202 OH (3

SCHEME6. Proposed mechanism for ITQ biogenesis

IS activated for reaction with oxygen by liganding to copper (Scheme 6A). Reaction with oxygen forms a peroxy intermediate (Scheme 6B), which can decompose to give dopa quinone (Scheme 6C). At this point, the mechanism differs from TPQ. The C2 position of dopa quinone can undergo nucleophilic attack by a lysyl side chain in the active site forming lysylquinol (Scheme 6D), which can then be oxidized to LTQ by molecular oxygen (Scheme 6E) in a mechanism very similar to the oxidative half-reaction of catalysis.

D. Biological Roles Increased activity of LOX is associated with a n u m b e r of fibrotic disorders (see Smith-Mungo and Kagan, 1998), and deficiencies in LOX activity have been implicated in Menkes' syndrome and type IX EhlersDanlos syndrome, among others (see Kivirikko, 1993). The identification of many LOX-associated diseases underscores the important physiological function of the enzyme. LOX is expressed as a prepropeptide with a secretion signal, consistent with its localization in the extracellular matrix (ECM). Following secretion, the 21 amino acid signal peptide is cleaved to yield the 50 kDa propeptide. The propeptide is then proteolytically cleaved, probably by procollagen-c-proteinase, to form the 32 kDa mature enzyme

170

JOANNE E. DOVE ANDJUDITH P. KLINMAN

(Smith-Mungo and Kagan, 1998). In the ECM, LOX plays an essential role in the cross-linking of procollagen and proelastin through the conversion of the e-amino group of lysyl residues to the aldehyde (SmithMungo and Kagan, 1998). LOX has also been identified as a tumor suppressor gene, which may result from a role in regulating cell proliferation (Contente et al., 1990; Kenyon et al., 1991). Another recent report has provided evidence of LOX activity within the nuclei of fibrogenic cells. Although the biological role of the enzyme in the nucleus is not known, suggestions have included an influence on chromatin structure or DNA transcriptional regulation (Li et al., 1997). Recently, a h u m a n enzyme with similarity to LOX, lysyl oxidase-like exzyme (LOXL), was identified (Kenyon et al., 1993; Kim et al., 1995). LOXL contains a secretion signal and has been localized to the ECM (I~lm et al., 1999). An endopeptidase cleavage site has been identified, suggesting that LOXL may also be proteolytically cleaved to form the mature enzyme. Further, LOXL is coexpressed with the type III collagen colIIIA1 gene (Kim et al., 1999), suggesting that it has a very similar role to LOX in processing collagen to form the ECM. A third m e m b e r of the emerging lysyl oxidase family, lysyl oxidaselike enzyme 2 (LOXL2) has been identified (Jourdan-Le Saux et al., 1999). Unlike the other two enzymes, LOXL2 does not have a hydrophobic signal sequence, suggesting that it might not be secreted from the cell. Using antibody detection, it has been localized both intra-and extracellularly (Jourdan-Le Saux et al., 1999). The role of this enzyme is not clear, but Csiszar and co-workers have suggested that LOXL2, rather than LOX, may be localized to the nucleus and involved in histone modification (Jourdan-Le Saux et al., 1999). The mammalian lysyl oxidases and lysyl oxidase-like enzymes have very similar i n t r o n / e x o n boundary structures and exon sizes within the conserved C-terminal domain (Kim et al., 1995; Jourdan-Le Saux et al., 1999), suggesting that they have all evolved from a c o m m o n ancestor. An alignment of the amino acid sequences (Table II) shows that the tyrosine and lysine modified to form LTQ are conserved among the family of LOX enzymes. As a result, LOXL and LOXL2 are expected to contain LTQ as a cofactor; however, purification and characterization of the proteins will be necessary for confirmation.

V~. PERSPECTIVE AND FUTURE DIRECTIONS

Despite the enormous progress that has been made in characterizing the catalytic mechanism of TPQ-containing enzymes, many questions

TPQ AND I,TQ

17 1

TAm.E I I

Sequence Aliuzment of Eysyl Oxidase Enzymes Enzyme

Sequence °

Rel~k~rence

Human LOX

VAEGHKASFCLEDT SCDYGYYRRYACTAH. TQG LSPGCND' _F_F_F_F_F_~I'RID AD CQW

H/imfil/iinen et el., 199 I

Hulnan LOXL

VAEGHKASFCLEDSTCDFGNLKRYACTS| f. TQGLSPGCYD~ADIDCQW VAEGHKASFCLEDTECEGDIQKNYECAN FGDQGITMGCWD~HDIDCQW

Kenyon el al., 1993

Human

LOXL2

.]ordan-Le Saux el al., 1999

'~The tyrosine and lysine proposed to be precursors of LTQ are underlined. Alignments were constructed using the progrmn pileup, one of the Genetics (:omputer (;roup ((;C(;) sequence analysis programs.

remain to be resolved. The nature of the protein structural elements that contribute to cofactor mobility during biogenesis, versus cofactor immobility during catalytic turnover, is particularly intriguing. Further site-specific mutagenesis of the residues contained within the "wall" of residues that resides behind the mature cofactor (cE Fig. 3C) may help us understand their control of the tyrosine and T P Q configurations required for biogenesis and turnover, respectively. In the literature of CA.Os, there is frequent mention of half of the sites reactivity. In fact, examination of X-ray structures indicates an intermingling of side chains from each sublmit of the dimer, both within the active site and at the dimer interface. Using this structural information as a guide, it may be possible to design experiments to resolve the longstanding controversy of half of the sites reactivity. One of the unexpected findings from the study of the oxidative halt: reaction of CAOs is the presence of a nonmetal dioxygen binding pocket. What are the protein structural requirements for such Oe binding and how does a hydrophobic site distinguish between, for exainple. 09 versus N~? While a working m o d e l has b e e n p r e s e n t e d for biogenesis (Scheme 4), u n a m b i g u o u s identification of the 350 nm species will require further spectroscopic probes. In the context of the postulated tyrosinate to c o p p e r charge transfer complex as the key intermediate in biogenesis, how does the binding of 02 initiate the inovement of tyrosine onto the copper? The electrophilicity of the c o p p e r center appears to be critical in modulating the spectral properties and reactivity of the charge transfer intermediate. Can fllrther probes of this intermediate, through site-specific mutagenesis, provide insight into the degree of electron delocalization from the

172

JOANNE E. DOVEANDJUDITH P. KLINMAN

tyrosinate to the copper ion and the impact of this delocalization on the rate of cofactor biogenesis? The LTQ-containing enzymes present a major challenge with regard to further characterization. The key to any substantive progress will be the availability of a reproducible, high-level expression system. To what extent is a cross-linked structure essential for lysyl oxidase activity and is it possible to create TPQ-lil~e cofactors at the active site of LOX mutants? There is also the relationship of the mechanism of TPQ and LTQ biogenesis to that of other modified tyrosine cofactors such as the crosslinked tyrosyl radical found in galactose oxidase. Will any of the principles gleaned thus far from studies of TPQ biogenesis be applicable to the biogenesis of cross-linked tyrosyl radicals? Finally, sorting out the increasing evidence for the important and varied physiological roles of the mammalian CAOs should occupy investigators for many years to come.

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