Multiple forms of liver microsomal flavin-containing monooxygenases: Complete covalent structure of form 2

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 290, No. 1, October, pp. 1033115, 1991

Multiple Forms of Liver Microsomal Flavin-Containing Monooxygenases: Complete Covalent Structure of Form 2’ Juris

0~01s of Biochemistry,

Department

University

of Connecticut

Health

Center,

Farmington,

Connecticut

06030

Received April 4, 1991, and in revised form June 7, 1991

Hepatic flavin-containing monooxygenases catalyze NADPH-dependent oxygenation of a wide variety of drugs that possess a nucleophilic heteroatom. Two forms of these enzymes (form 1 and 2) have been isolated from rabbit liver microsomes and partially characterized (Ozols, J., 1989, Biochem. Biophys. Res. Commun. 163, 49-55). The complete amino acid sequence of form 2 is presented here. Sequence determination was achieved by pulsed liquid-phase and solid-phase sequencing of 40 peptides generated by chemical and enzymatic cleavages, including CNBr cleavage of tryptophanyl residues. Form 2 monooxygenase contains 533 amino acid residues and has a molecular weight of 60,089. The COOH terminus of this enzyme is very hydrophobic and presumably functions to anchor the protein to the membrane. Form 2 is readily degraded, since a form lacking residues 1 to 278 and a form without the COOH-terminal segment were also isolated from solubilized membrane preparations. The amino acid sequence of form 2 is 52% identical to that of form 1 and shows 55% identity to the sequence of rabbit lung monooxygenase derived from the cDNA data. The putative FAD and NADP binding segments around residues 9 and 190 are conserved in all three forms. Three variable segments can also be identified in these isoforms. These are residues 308 to 321, residues 408 to 421, and the membrane binding domain, residues 505 to 533. A comparison of the presently limited amino acid sequence data of flavin-containing monooxygenases (FMOs) implies that a particular FM0 in different mammalian species may be very similar, but isozymes within a species may exhibit more extensive variability with respect to homology and catalytic activity. This study documents the structural diversity of a second hepatic FM0 from rabbit liver and establishes this class of drugmetabolizing enzymes as a family of related proteins. 0 1991

Academic

0003.9861/91

Press.

Inc.

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Studies from several laboratories have shown that mammalian microsomal membranes contain flavoproteins that catalyze NADPH-dependent heteroatom oxygenation of xenobiotics, including antipsychotic and narcotic drugs2 (1). In an earlier communication, I reported the isolation of two structurally related flavin-containing monooxygenases (FM0)3 from rabbit liver microsomal membranes (2). Partial amino acid sequence analysis indicated that these two forms, designated form 1 and 2, were derived from related but distinct genes (2). More recently, I reported the complete amino acid sequence of form 1 (3). This form showed about 90% identity to the recently reported cDNA sequence encoding the enzyme from hog liver microsomes (4). Sequences derived from cDNAs coding for the flavin-containing monooxygenases expressed in rabbit lung and liver have also appeared (5). Very recently, Yamada et al. (6) report the isolation of two forms of FMOs from liver microsomes of guinea pigs. These forms appear to be similar to forms 1 and 2 of the rabbit liver. While the NH2 terminus of the 56-kDa form was blocked, the NH,-terminal sequence of 19 residues of the 54-kDa form showed 90% identity to the rabbit form 2. In an effort to increase our understanding of this group of enzymes, I report here the covalent structure of form

r This work was supported by United States Public Health Service Grant GM26351. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ Portions of this paper (including Experimental Procedure, Figs. S1 to S-25) are presented in miniprint at the end of this paper. 3 Abbreviations used: HPLC, high performance liquid chromatography; HFBA, heptafluorobutyric acid; GNCl, guanidine hydrochloride; PTH, phenylthiohydantoin; TFA, trifluoroacetic acid, FMO, flavin-containing monooxygenases; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 103

104 GUY LYs

LYs

Val

Ala

Ile

Ile

Gly

Ala

10 Gly

Ile

SW

Gly

JURIS

OZOLS

Leu

Ser

Ala

Ile

Arg

20 Cys LW

SW

Glu

Glu

-

Glu

Met

Ser

Asp

Asp

Ile

Gly

Gly

Leu

40 Trp

Ser

Thr

Ser

Asp

His

Ala

Glu

Glu

Gly

Glu

Pro

Thr

Cys

30 phe

Gln

Ser

Val

Phe Thr

60 Asn

Gln

Glu

Tyr

90 Ile

Asp

Phe

Ser

120 Val

Gly

His

His

150 Val

Gly

180 Ile

R-2 50 Arg Ala

Ser

Ile

Tyr

R-3

CB-2 Lys

Glu

Met

Met

Cys

Phe

Pro

70 Asp Phe

Phe

Pro

Phe

Pro

Pro

Asp

Phe

Pro

Asn

80 Asn kt

Ala

Arg

Glu

Lys

Asn

Leu

100 Leu

His

Asn

Ser

LYS Leu

v

T-6

I-

Thr

Phe

Leu

-

I Ser

Lys

Gly

Lys

Tyr

Ile

Gln

Phe

Lys

Thr

Leu

Val

110 Ser

Ser

CB-4

Ile

Lys

Lys

His

Pro I

R-4

t

Thr

Gly

Gln

Trp

-

Tyr

m-4;

Val

Ala

Thr

Cys

130 Arg

Asn

Gly

Lys

Lys

Glu

Pm

Asn

-

Ala

140 Phe Asp

Val

Ala

Val

Met

D-2

Leu

Pro

Lys

Asp

Ser

Phe

160 Pro

Gly

Leu

Lys

Gly

Lys

Arg

Lys

His

Val

CB-5;

Phe

Lys

Gly

Lys

Ser

170 Phe

Arg

Gln

Arg

CB-5;

Ile

Ser

-

Ser

Leu

Val

Ile

190 Gly

Phe

Leu

Lys

C&5-

Leu

Gly

Asn

Ser

Glu

Tyr

Lys

Gly

Glu

Asp

Ile

Ala

200 Thr

Pro

230 Trp

Glu

Leu

Ser

Ser

His

Thr

Ala

Asn

Leu

Asn

Gly

Gly

Ser

Trp

Val

Ser

Arg

T-18 t-Leu

Pro

Thr

Ala

250 Ile

Ser

Val

I CB-6

Asp

Trp

Thr

L&u

Arg

Lys

280 Glu

Thr

310 Glu

Pro

Val

Trp

Asp

Asp

Gly

Tyr

Asp

F!et

Leu

Tyr

Val

Thr

Lys

Glu

Phe

Lys

Glu

Phe

Thr

Ser

Thr

CB-9, Gly

Tyr

Trp

Tyr

Val

Lys

Gln

CB-9,

Ala

Gln

Leu

-

Ala

Glu

Lys

Pro

IO; E-6

Thr

Met

261) Asn Ala

Lys

Phe

Lys

His

Glu

Gln

Val

Lys

Gly

Phe Gly

Lys

Trp

Thr

Phe

340 Leu

Asp

Ala

Pro

Ala

290 Arg

Ile

Leu

CYS Gly

Thr

Val

Ser

Ile

Lys

Phe

Glu

Aso

Gly

Thr

320 Val

Phe

Glu

Ala

Asp

Ser

Val

Ile

330 Phe Ala

Asn

350 Asn

Trp

Ile

Asp

Ser

Ile

Ile

Lys

Phe

Leu

Met

Ala

370 Val

Ile

Gly

Leu

Ser

Thr

Pro

Gly

Ala

Arg

VCB-14; -R-l5 bR-I6 Ala -

Leu

Phe CB-I5

Ser

Thr

Cys

400 Thr

Leu

Pro

Ile

Gln

430 Thr

Asp

Tyr

Val

Gln

Ser

Pro

Val

Lys

Asp

Ile

Asn

Tyr

Met

E-3

Asp

Pro

Arg

Leu

460 Ala

Leu

490 Thr

Leu

520 Leu

Leu

Glu

Lys

Val

Thr

Gln

Glu

Leu

Phe

Lys

Gly

Leu

Gly

Ala

380 Ala

Ile

Pro

Thr

Thr

Asp

Leu

Gln

Ala

Ile

Leu

Trp

Ile

360 Pm

Phe

T-30

-

Ala

Arg

Met

Asp

Met

Asn

410 Asp

Ile

His

Glu

Glu

Phe Phe CB-14;

Gly

Pro DP-2

Lys

Met

Gly

Thr

Gln

Trp

Ala

Ile

___ R-18

ASD Arg

b---

Ala

Val

Ser

Leu

Lys

CB-14;W-5-

Leu

Leu

Ile

Lys

Leu

420 Lys

-RR-t3;E-2t----B-13-

Leu

440 Ala

Ser

470 Pro

Met

500 Lys

Ala

530 Val

Ser

Phe

Val

290 Trp

t--

Ile

Gly

Val

Lys

Leu

Asn

Ile

450 Pro

Leu

Val

Gly

Pro

Gly

480 Lys

m-14 Cys

Tyr

Gln

Phe Arq

-

W-4 ___

Pro

Glu

E-6 -

CB-II

t

Tro

Ile

+

CB-I3 Leu

300 Pro

R-12; D-8 ~

-RR-13; -

270 Leu

Leu

-CB-I2 Glu

Ser

Asp

t Ile

Tyr

Aso

R-13

Thr

Asn

CB-8

~

T-30

Ala

240 Thr

T-27

Tyr

Pro

Gln

10

Pro

Phe

CB-7

+CB-9,tO; Tyr

210 Val

I +

IO Gly

Arg

R-9

Phe Asn

CB-9, Val

Val

I- R-IO; E-2 -

I Asn

Gln

I- CB-5; D-7220 Met

+ Pro

Glu

R-7

V D-7 ~ Asn

Pro

R-6 + CB-5; D-3 -

R-IO

Met

Glu

D-3

Arg

CB-5;

Ser

-

D-2

I

-

Cys

R-5

CB-5

Phe

Ile

-

Tyr

Thr

Pro

Ala

R-17

Thr

Leu

Arp

Val

Ala

Val

CB-15

Phe.

Gly

R-15His

Leu

R-18

Gln

Lys

510 Pro

COVALENT

STRUCTURE

OF LIVER

MICROSOMAL

MONOOXYGENASE

FORM

2

105

21)). The latter gave better recoveries, but gave less efficient resolution of peptides than the larger column. Tryptic cleavage of succinylated form 2 protein was carried out with about 6 nmol of alkylated protein. All of the expected peptides, except R-12, were isolated from this digest by HPLC. Peptide R-12 (residues 291 to 389) eluted in the void volume of the gel filtration column, in partially MATERIALS AND METHODS purified form, but was lost upon HPLC fractionation. The NH2 terminus of peptide R-16 was blocked, but digestion Materials. Detergents, enzyme substrates, cofactors, and chemicals, unless stated otherwise, were obtained from Sigma. [l-‘4C]iodoacetamide with pyroglutamyl aminopeptidase removed the blocking was from New England Nuclear. DEAE-cellulose (DE-52) was from group, and eight cycles of Edman degradation established Whatman Chemical Separations, and hydroxyapatite-agarose (HA-Ulits sequence. The HPLC isolation profiles of peptides tragel) was a product of IBF Biotechnics. TFA, HFBA, CNBr, GnCl, used for the proof of this sequence are shown in Figs. and urea were obtained from Pierce Chemical. Solvents for HPLC and gel filtrations were from Burdick and Jackson. Trypsin, a-chymotrypsin, S-l to S-6. and pepsin were obtained from Worthington. Stuphylococc~saureus Cleavage of form 2 at methionyl residues gave another protease, endoprotease Asp-N, and pyroglutamyl amino peptidase were set of peptides (Figs. S-7 to S-13). At residue 278, both obtained from Boehringer-Mannheim. Liver microsomal flavin-conMet and Arg were observed and at residue 404 both Val taining monooxygenase form 2 was isolated from adult New Zealand and Met were observed. Arg was the predominant residue male rabbits as previously described (2, 15). at 278 and Val at 404. Fragments obtained by CNBr and Methods. Routine amino acid analyses were carried out with 6 N HCl in the gas phase at 150°C for 1 h (16) using a Beckman Model 6300 tryptic digestion were further cleaved by endopeptidase amino acid analyzer equipped with a ninhydrin detector. The COOHAsp-N (Figs. S-14 to S-20). Cleavage at the NH2 side at terminal peptides of form 2 protein were also hydrolyzed for 12, 24, and both Asp and Glu residues was observed with this enzyme. 92 h with 6 N HCl in evacuated sealed tubes at 107°C. Acid cleavage at the Asp-Pro bond in peptide CB-14 proPeptide mixtures, unless stated otherwise, were first separated using a 2.5 X lO@cm or a 1.5 X loo-cm column of LH60 Sephadex equilibrated vided the necessary overlap for peptides CB-14 and Rwith formic acidethanol (3:7) as the solvent as previously described (6). 15. Cleavage of Trp residues in CB-14 by CNBr was esThe peptide mixtures from the gel filtration column were further resolved sential to establish the critical overlap for peptides R-16 by using reverse phase HPLC. The methodology has been detailed in (16). and R-17 (Fig. S-17). Reverse phase columns used for separations include Vydac C4 (15 Tryptic cleavage of the native protein (3 nmol) followed X 0.46 cm) and Waters Associates C18, PBondapac (30 X 0.39 cm). Peptide mixtures were dissolved in 88% formic acid prior to the injection. by carboxymethylation, gel filtration of the digest, and The type of solvent used in these separations is indicated in the legend HPLC analysis of individual fractions gave essentially all of each chromatogram. of the expected tryptic fragments that were larger than Sequence analysis of peptides was carried out on an Applied Biosysfive residues (Fig. S-21). Only peptides T-6, -18, -27, -30, terns Model 470A, with a pulsed liquid-phase solvent delivery system, equipped with a Model 120A PTH analyzer according to manufacturers and -42 were essential for the completion of the sequence instructions. Solid phase sequencing was performed with a Milligen/ of form 2 (Table SI and Figs. S-22 to S-25). The results Biosearch 6600 ProSequencer system as described in the miniprint secof automated sequence analysis of peptides used to deduce tion. Monooxygenase activity was measured by monitoring methimazolethe complete primary structure of form 2 are given in dependent NADPH oxidation as described in (2). Table SI.

2 monooxygenase and compare its structure to form 1 and the cDNA-derived sequences of the enzymes present in the liver and lung microsomes. The covalent structure of form 2 enzyme is the first report of an amino acid sequence of a hepatic FM0 isoform.

RESULTS

Sequence of form 2 monooxygenase. The general strategy of sequence analysis of form 2 enzyme involved the reduction and carboxyamidomethylation of the protein followed by chemical and enzymatic cleavages. Resolution of the digests was achieved by fractionation on an LH-60 column followed by HPLC of individual fractions (7). Two sizes of LH-60 columns were used (2.5 X 100 cm (Fig. S-l) and 1.5 X 95 cm (Figs. S-l, 8, and

Determination of sequence of the COOH terminus. Peptides corresponding to the COOH terminus of form 2 protein were isolated from tryptic digests of succinylated protein and of the intact form. The HPLC elution profile and the amino acid composition of these peptides were identical (Figs. S-3 and S-25). A peptide (CB15) with an HPLC elution profile similar to peptides R-18 and T-42, but having three additional residues, was isolated from CNBr digests of form 2 (Fig. S-8). Conventional automated sequence analysis of each of these pep-

FIG. 1. Covalent structure of liver microsomal flavin-containing monooxygenase form 2. Peptides obtained by tryptic cleavage of intact or succinylated protein are denoted by (T-) and (R-), respectively. Peptides obtained by CNBr cleavage are denoted by (CB-). Subcleavage of peptides by endoprotease Asp-N or Stuphylococcusaureus protease is denoted by (D-) and (E-), respectively. Prefix (DP-) marks peptides obtained by cleavage with formic acid, and (W-) denotes peptides obtained by cleavage at tryptophanyl bonds by CNBr. Residues identified by automated sequence analysis are indicated by a solid line. The covalent structure of peptide R-18 was determined by solid phase sequencing methodology. The sequence of residues 1 to 32 and 222 to 263 was reported previously (2). Residues 278 and 404 were replaced by methionyl residues in digests of some form 2 preparations. Residues 253 and 262 in this sequence are correctly reported as Trp and Lys rather than Met and Trp as erroneously reported earlier (2).

106

JURIS

OZOLS

l*Ala 2 Gly L Ala

Lys Lys Lys

Arg Lys Lys

Val Val Val

Ala Ala Ala

Ile Ile Val

Val lle Ile

Gly Gly Gly

Ala Ala Ala

10 Glv Yal Gly Ile Gly Val

Ser Ser Ser

Gly Gly Gly

Leu Leu Leu

Ala Ala Ile

Ser Ser Ser

Ile lle Leu

Lys Ara Lys

20 Ser Cys Leu Ser Cys Leu CYS Cys Val

Glu Glu Glu

Arg Met Arg

Ser Ser Thr

Asp Asp Glu

Asp Asp Asp

Leu Ile lle

Gly Gly Gly

Cly Gly Gly

Leu Leu Leu

40 Trp Arq Trp Lys Trp Arg

Phe Phe Phe

Thr Ser Lys

Glu Asp Glu

His His Asn

Val Ala Val

Glu Glu Glu

Glu Glu Asp

Clv Gly Gly

Ser Ser Thr

Cys Ser Ser

Lys Lys Lys

Glu Glu Glu

!let Net llet

Ser Met Ser

Cys Cys Cys

Tyr Phe Phe

Ser Pro Ser

7u Asp Phe Pro Asp Phe Pro Asp Phe Pro

Phe Phe Met

Pro Pro Pro

Glu Pro Glu

Asp Asn Asp

Tyr Phe Phe

Pro Pro Pro

Lys Thr Arp

Met Thr

Ile

Tyr Ala Pne Ala Phe Ala

Asp Arq Lys

Arg Glu Lys

Phe Asp Lys Asn Phe Asp

Leu Leu Leu

100 Leu Leu Leu

Lys Lys Lys

Glu Tyr Tyr

lle lle Ile

Gln Gln Gln

Phe Lys Phe Lys Phe Gln

Thr Thr Thr

Ser Thr Ser

Gly Gly Gly

Gln Gln Gin

Trp Trp Trp

Lys Tyr Glu

Val Val Val

Val Ala Val

Thr Thr Thr

Leu Cys Gln

130 His Arg Ser

Glu Asn Asn

Gly Gly Ser

Lys Lys Lys

Gln Lys Gln

Glu Glu Gln

Ser Thr Ser

Asn Tyr Leu

Pro Pro Pro

His Asn Asn

Leu Leu Ile

Pro Pro Pro

Leu Lys Leu

Gly Asp Lys

Cvs Ser Ser

Phe Phe Phe

160 Pro Pro Pro

Gly Gly Gly

lle Leu Ile

Lys Lys Glu

Thr His Lys

Phe Phe Phe

Phe Lys Phe Lys Leu Glu

Asp Gly Gly

Lys Lys Lys

Arg Arg Arg

Val Val

Ile

Leu Leu Leu

Val Val Val

Val lle Ile

190 Gly Gly Gly

Ilet Leu lle

Gly Gly Gly

Asn Asn Asn

SW Ser Ser

Gly Gly Ala

Leu

lle Ile

Ser Ser Ser

Thr Ser Thr

Thr Arg Arg

Gly Ser Lys

Gly Gly Gly

Ala Ser Ser

Trp Trp Trp

Val Val Val

220 lle Met Met

Ser Ser Ser

Arg Arg Arg

Val Val lle

Phe Phe Met

Ile Leu Leu

Arg Lys Arg

Asn Asn Asn

Ser Asn Val

Leu Leu Leu

Pro Pro Pro

Thr Thr Arg

Pro Ala Met

250 Ile lle lle

Val Ser Val

Thr Asp Lys

Trp Trp Trp

Val Met Ala

Pro Pro Pro

Lys Leu Glu

Asp Asn Asn

Arg Gly Lys

lle Thr Tyr

Gln Leu Leu

Met Arg Met

Lys Lys Lys

280 Glu Glu Glu

Pro Pro Pro

Val Val Val

Ser Am Arg

Ile Val Val

Lys Lys Lys

Glu Glu Glu

Val Phe Leu

Lys Lys Thr

Glu Glu Glu

Asn Ser Phe Thr Ser Ala

310 Val Glu Ala

Val Thr lle

Phe Gly Ser Ala Phe Glu

Thr Thr Thr

Gly Gly Gly

Tyr Tyr Tyr

Thr Gly Thr

Phe Ala Tyr Ala Phe Ala

Phe Tyr Phe

Pro Pro Pro

340 Ser Leu Phe Leu Phe Leu

Asp Asp Glu

Glu Asp Glu

Tvr Trn Leu I,.

Tvc --Ser I,-

Ser Ser Ser

Leu lle lle

Tyr Tyr Tyr

Lys Gln Gln

Ser Ser Ser

Val Val Val

Val Phe lle

Ser Thr Thr

60 Asn Asn Asn

Asn Asn Asn

60 Tyr Val Asn Met Phe Leu

Pro His His

Asn Asn Asn

Ser Ser Ser

Gln Lys Lys

Phe Leu Leu Gln Leu Leu

Asp Glu Glu

Tyr Tyr Tyr

90 Leu lle Phe

Thr Leu Thr

Val Val Val

110 Phe Ser lle

Ser Ser Ser

lie Ile Val

Thr Lys Lys

Lys Lys Lys

Glu His Ara

Gln Pro Pro

Asp Asp Asp

Phe Asn Phe Ser Phe Ala

120 Val Val Ser

Ala Ala Ala

lie Val Val

140 Phe Asp Phe Asp Phe Asp

Ala Ala Ala

Val Val Val

Wet Met Met

Val lle Val

Cys Cys Cys

Thr Ser Ser

Gly Gly Gly

Phe His His

Leu His His

150 Thr Val lle

Lys Lys Lys

Gly Gly Gly

Gln Lys Gln

Tyr Ser Tyr

170 Phe Phe Phe

His Arq His

Ser Gln Ser

Arg Arg Are

Gln Glu Gln

Tyr Tyr Tyr

Lys Lys Lys

His Glu His

Pro Pro Pro

Asp Gly Ala

180 lle lle Gly

Thr Glu Ser

Asp Asp Asp

lle lle lle

Ala Ala Ala

200 Val Thr Val

Glu Glu Glu

Ala Leu Leu

Ser Ser Ser

His His Lys

Val Thr Lys

Ala Ala Ala

Lys Glu Ala

Lys Gln Gln

Val Val Val

210 Phe Val Tyr

Phe Asp Trp Asp Ser Glu

Ser Gly Asp Gly ASD Gly

Tyr Tyr Tyr

Pro Pro Pro

230 Tro Trp Trp

Asp Asp Asp

Met Met Met

Val Leu Val

Phe Tyr Phe

Thr Val His

Thr Thr Thr

Arg Arg Arg

Phe Phe Phe

240 Gln Asn Gln Thr Set- Ser

Leu Trp Met

Val Tyr Met

Ala Val Glu

Lys Lys Gin

Lys Gln Gln

Met Met flet

260 Asn Asn Asn

Ser Ala Arg

Trn Lys Trp

Phe Phe Phe

Asn Lys Asn

His His His

Ala Glu Glu

Asn Asn Asn

Tyr Tyr Tyr

Gly Ser Gly

Leu Asn Asp Phe Asn Asp Leu Asn Asp

Glu Asp Asp

Leu Leu Leu

Pro Pro Pro

Gly Ala Ser

Arrl Arg Arg

lle lle lle

Ile Leu Leu

Thr Cys Tyr

Gly Gly Gly

Lys Thr Thr

Val Val lle

Phe Ser Lys

lle lle Val

Arg Lys Lys

Asn lle Asp

Ala His Phe Glu Glv ---

Asn Asp ---

Thr Gly ---

Pro Thr Thr

320 Ser Val Val

Glu Glu Phe Glu Glu Glu

Pro Ala

lle

lle lle

Val Ser Val

lle Val lle

Val

ASD

Asp Asp Aso

Phe Ala Phe Ala Phe Ala

Val Ile Leu

Val

Val Ser

Glu Glu Glu

Asp Asn Asp

Gly Asn Asn

Gln Lys Met

Ala Val Val

Ser Thr Ser

Leu Leu Leu

Tyr Phe Tyr

Lys Lys Lys

Tyr Gly Tyr

Leu lle Phe

Pro Pro Pro

Thr Thr Thr

Gly Thr Val

Glu Aso Glu

Thr Leu Leu

Val

Asn His

Arg Lys Arg

Lys Met Asn

290

lle Val

Lys Lys Lys

lle

lle Val

360

Ile lle Met

Phe Phe Phe

Pro Pro Pro

Gln Gln Gln

Ala Ala Ala

Arg Arc Arg

390 Tyr Tro Trp

Glu Gly Glu

Asn Thr Asn

Lys Lys Arg

His Leu lle

Asn Lys Ala

Ala Val Ala

Lys Lys Lys

Pro Leu Pro

450 Asn Asn Asp

Val Thr Leu

400 Lys Cys Thr Cys Ser

Leu Leu Leu

Pro Pro Pro

Pro Pro Ser

Thr Val Lys

Val Asp Thr

Met Met Met

lle Met Met

Lvs Asn Ala

410 Glu Aso Asp

lle

lle

Glu Glu Lys

Ivz Glu __ Gln

Ala T hr Lys

430 Leu lle Leu

Asp Asp Asp

440 Asp Glu Glu

Leu Leu Leu

Leu Ala Ala

Thr Ser Leu

Ser Phe Glu

Ile lle lle

Asn Gly Gly

Leu Leu Leu

Ala Ala Ala

Leu Leu Val

Thr Glu Lys

Met Val Leu

Phe Phe Tyr

Phe Gly Phe Gly Phe Gly

Pro Pro Pro

470 Tyr Cys Cys

Ser Ser Asn

Pro Pro Ser

Tyr Tyr Tyr

Gln Gln Gln

Phe Phe Tyr

Arg Arg Arg

Leu Leu Leu

Thr Val Val

Gly Gly Gly

Met Thr Leu Thr Phe Thr

Gln Gln Gln

Trp Trp Lys

Asp Asp Gln

Am Arq Arq

Thr Ser lle

Phe Leu Leu

Lys Lys Lys

500 Val Pro Pro

Thr Met Leu

Lys Lys Lys

Thr Thr Thr

Arq Arg Arq

Ile Ala Thr

Val Val Leu

Gln Gly Lys

Glu His Ala

Ser Leu Ser

Leu Ala Leu

Ala Leu lle Ala Phe Ala

Leu Val Leu

Val Leu Val

Ser Leu Leu

Val lle Ala

530 Phe Leu Ala Ala Phe Lcu

lle Phe Val Leu Phe Gln

Leu Val Leu

(Glu,Ser). Phe Gln Trp

Phe

-,-

lle

,.

Gln Gln Gln

Leu Ile Leu

Phe Ser Pro Trp Val Ser

Ser Leu Phe

Leu Gly Thr Phe Leu Thr Leu Phe Lys

Asp Asp Asp

Pro Pro Pro

460 Leu Arg Lys

Gly Gly Gly

Lys Lys Gln

Trp Trp Trp

Lys Pro Glu

Gly Gly Gly

Ala Ala Ala

Arq Arg Arg

Asn Gln Asn

Ala Ala Ala

490 lle lle Ile

Ser Lys Asn

Pro Phe Pro Ala Phe Pro

Glu Leu Val

Ser Phe Ser

Leu Ser Phe

Leu Pro Leu

Lys Glu Leu

Leu Leu Lys

520 Phe Ala Trp Leu Phe Leu

Ala Thr Thr

Val Leu Gly

Asp Asp Asn

Tyr Tyr Tyr

Cys Lys Glu

Ile lle

lle

Thr Asn Asp

Tyr Tyr Tyr

lle Met Leu

Pro Pro Arg 330

350 Ser Ser Ser

270 Leu Leu Leu 300

Ser Ala Ser

Lys Lys Lys

I.,-

50 Are Ala Arg Ala Arg Ala

Gly ,ly G Gly

Phe Ile Phe

L..”

30 Phe Phe Phe

lull __" ___ IN Leu

Gln Gln Arg

lcllc Tvz I YC- --_, Glu Ser

Cys Cys Cys

Pm._ SW Pro

Val Ala Thr

V.,

Thr Thr Thr

110 L._ Ivc ..,.. _., Val,. Gin 11 e Gln

Thr Ala Ala

r.1~ Thr Phn_ T,lv - , LeL I Phe Gly

Pro Pro Pro

,-,, cc” Leu Leu

CT..Th.. l,?,, “0” *,. rl” ,111 L-G” Pro Thr Met Ala Ser Thr Phe Ala

!llC

Lys Glu Glu

C,$, Y’J Gly Gly

I,,* LJ> Lys Lys

“‘J

Leu Leu Leu

,,,, IIC Ile Leu

I.... LIZ” Cl" “ill Leu Glu Leu Glu

r.11, ~0

Gly Gly Gly

380 Met Ala Ile

,a:“13 Gln Gln

Gly Gly Gly

Glu Glu Glu

370 \,., .“I Val Cys

"1_ n,a Pro Pro

Val Val Val

Glu Glu Asp

420

lle

480 Pro Pro Pro 510 Ser Gln Ser

FIG. 2. Comparison of amino acid sequences of rabbit liver microsomal forms 1 and 2, and the rabbit lung flavin-containing monooxygenases. The sequence of the lung enzyme was derived from the cDNA structure (5). The NH, terminus of form 1 is acetylated (3). The NHz-terminal methionyl residue has been deleted from the lung enzyme. In form 1, residues 252 and 253 are correctly reported as Thr and Trp rather than Ser and Thr as erroneouslv reported earlier (2).

“-\TT.

r

I,““XLl!,l~l

-.lm

“m-T,nmTT--

blJx”LlU~l3

-r.

ur

T

r.,nn

LIVl!,K

lull = ““,ROSOMAL

tides was unsuccessful. After cycle 14, the yield of the PTH derivative was below the limit of detection. Presumably, their extreme hydrophobicity led to a washout from the reaction cell of the sequencer. Coupling peptide R-18 or T-42 to an arylamine membrane (Sequelon-AA TM) using (1-ethyl-3-dimethylaminopropyl) carbodiimide as a coupling agent and sequencing the membrane with the MiliGen/Biosearch 6600 sequencing system overcame the washout problem and provided definitive assignment of all the residues in these peptides (Fig. S-I). The proof for the sequence of residues 1 to 32 and 222 to 263 was reported previously (2). DISCUSSION

The amino acid sequence of FM0 form 2 is summarized in Fig. 1. The protein is a polypeptide consisting of 533 residues. Peptides obtained from CNBr and tryptic digestion were characterized extensively. Most residues were confirmed by at least two sequencer runs. Unlike form 1, the carboxyamidomethylated form 2 protein was refractory toward S. aureus protease digestion. Except for the Glu residues at positions 322 and 348, only partial cleavages occurred at other glutamyl residues, resulting in a digest that was too complex to be resolved. Particularly useful fragmentation was obtained by the cleavage of tryptophanyl residues by excess CNBr in the presence of heptafluorobutyric acid (8). Cleavage of peptide CB-14 by this method established the essential overlap for peptides T-15, -16, and -17. The cleavage of Trp residues by CNBr is very simple to use and does not require reagents that are sensitive to storage or require the addition of difficult to remove phenol or indole derivatives. This cleavage procedure involves only a simple evaporation step to remove reactants and products prior to HPLC separation or sequence analysis. The structure of the COOH-terminal peptide could not be achieved without the automated micro-solid-phase sequencing methodology. The conventional sequencing of these peptides, immobilized on a Polybrene-coated fiberglass matrix, was not successful for more than 10 cycles (Table S-I). Presumably, the peptide was lost during the extraction steps of the automated Edman degradation procedure. In contrast, solid-phase sequence analysis of this peptide was quite successful in establishing its complete sequence (Table S-I). Three Asn-X-Ser/Thr sequences (at Asn 60, 267, and 274), which are glycosylated in microsomal proteins having such sequences oriented toward the luminal side of the membrane (9), are not glycosylated in form 2 monooxygenase. Similarly, the Asn-X-Ser sequences in liver microsomal cytochromes bf, (Asn 20 and 105) are not glycosylated, since they do not extend into the luminal side of the membrane, but are oriented toward the cytosolic side or are partially buried in the lipid bilayer (10). The form 2 enzyme has several sites that are particularly sensitive toward proteolysis. In some preparations a form with a slightly lower molecular weight than form

MONOOXYGENASE

FORM

2

107

2, but with 20 NH,-terminal residues identical to form 2, was identified by SDS-PAGE, electroblotting, and sequence analysis. Presumably, this form results from cleavage of about 30 residues from the COOH terminus. Another form, having a subunit molecular weight of approximately 25,000, with 20 NH,-terminal residues identical to residues 279 to 299 was also identified by SDSPAGE and electroblotting of partially purified form 2 preparations. These findings imply that these sites may exist as a surface segment in the tertiary structure of this FM0 and may be sites for protein degradation in uiuo. Of interest is that at residue 278, both Arg and Met were observed, but Arg was the predominant residue. Whether a site having an amino acid substitution results in a conformation that is more susceptible to proteases than the site containing the predominant residue remains to be determined. The comparison of the amino acid sequences of forms 1 and 2 is shown in Fig. 2. Residues at positions 425 and 427 are deleted in form 2, and an extra residue is inserted at position 535. The two forms share 52% identical residues. Form 2 has five additional basic and five additional acidic residues, so that the net charge difference between the two forms is conserved. While clusters of short segments of nonidentical residues are scattered throughout the entire sequence, two larger stretches of nonidentical residues are evident at residues 308 to 321 and the membrane-binding domain. Recently, sequences derived from cDNA structure coding for FMOs expressed in hog liver (4) and rabbit lung and liver have also appeared (5). The cDNA sequence of the liver enzyme, except for 10 amino acid assignments, is identical to that of form 1 (Table S-II). Six of the variant residues occur in the positions corresponding to a specific enzymatic or chemical cleavage site. The identity between the hog liver and the rabbit liver form 1 is about 90%. The lung enzyme, however, shows 55% identity to the form 1 sequence and 54% identity with the form 2 sequence (Fig. 2). When forms 1 and 2 are compared with the lung enzyme, only 42% of the residues are identical among the three forms. The proposed FAD and NADP pyrophosphate-binding domains around residues 9 and 190 are conserved in all three forms. Sequence comparison of all three rabbit FM0 forms identifies a third segment containing variable residues (residues 408 to 421). Whether these segments are responsible for the individual substrate specificities of these enzymes or represent “variable” domains of the monooxygenase molecules remains to be determined. The two hepatic FM0 sequences are unusual in that form 1 from rabbit (3) and hog (18) liver have an N-acetyl Ala at the NH, terminus, whereas the form 2 isozyme starts with a free glycyl residue. The NH2 terminus of the cDNA-derived lung FM0 is methionine, and the nature of the NH2 terminus of the expressed lung enzyme is not known (5). It has been proposed that N-acetylation may affect the biological function and/or protein stability, and

108

JURIS

that lack of acetylation can directly affect protein structure making it more unstable to denaturing conditions or more susceptible to proteolytic modification (20). The observation that form 2 FM0 is less stable toward storage than the N-acetylated form 1 is in agreement with this hypothesis. Furthermore, the observation that form 2 has several sites that are sensitive toward proteolysis supports the N-acetylation/stabilization hypothesis. While the cDNA sequence of the hog and rabbit lung enzymes indicate a methionyl residue at the NH2 terminus and the absence of a signal peptide sequence (4, 5), it remains to be determined whether the form 2 gene encodes a leader sequence. The second prominent feature of the monooxygenase sequences is the complete absence of polar residues at their COOH termini. Other microsomal membrane proteins sequenced to date terminate with a short stretch of negatively or positively charged residues. The significance of this structural feature for the degradation or the topology of this group of proteins remains to be elucidated. Recently, Hlavica et al. (11) reported that polyclonal antibodies to a synthetic 14-amino-acid peptide, residues 466 to 479 of form 1, cross-reacted with liver microsomes and with the partially purified liver enzyme, but did not recognize the protein in intact lung microsomes or the purified lung enzyme. This implies that the critical epitope may be exposed to the surface of the FM0 molecule when in the membrane-bound state in rho. Failure of this antibody to recognize the lung enzyme may be due to the amino acid sequence differences between the liver and the lung forms. Residues 466 to 479 of form 1 show only 57% identity when compared with the corresponding region of the lung form, whereas this segment in forms 1 and 2 shares 86% identical residues. These results are in agreement with studies that report that antibodies directed against the intact rabbit lung enzyme failed to demonstrate cross-reactivity with the hepatic enzyme from rabbits (12,13). The most intriguing questions regarding the microsomal FMOs cannot be answered by this study, since this is only the third reported sequence for this group of proteins. The recent report, that a FM0 form related to form 2 enzyme can be isolated from liver microsomes of guinea pigs (6), clearly indicates that form 2 enzyme may be more prevalent than previously suspected. Notwithstanding, certain conclusions concerning the biology of microsomal FMOs may be postulated from this study. Since the ammo acid sequence of rabbit liver FM0 form 1 is some 90% identical to that of form 1 from hog liver (4, 5), the amino acid sequence of form 1 in other mammalian species will be very much identical. From past sequence comparison studies, such a conclusion is now well supported. More importantly, since form 1 from other species is structurally very identical, the results of the extensive studies on substrate specificity, reported with the hog liver form (1)) can now be extrapolated to form 1 in other species. The sequence of rabbit lung enzyme is only 55% similar to the rabbit liver form 1. Rabbit lung

OZOLS

FM0 differs significantly in the substrate specificity from the hog liver form 1 (1, 12-14). For example, unlike liver form 1, the rabbit lung enzyme cannot distinguish primary from secondary or tertiary amines (1). On the other hand, many of the better substrates for liver form 1 (e.g., chlorpromazine, imipramine, benzphetamine) show no activity with the lung enzymes (21). Contrary to the hog and rabbit liver form 1, the amino acid sequence of rabbit liver form 2 is only 54% identical to that of rabbit form 1 and shows only 55% identity to the rabbit lung enzyme. The catalytic properties of form 2 FM0 will be elucidated in our next study. ACKNOWLEDGMENTS I am grateful to George Korza for his outstanding technical assistance and to Dr. Vijay Kumar for his skillful isolation and solubilization of microsomal membranes. I also thank Dr. David W. Andrews (MilliGen/ Biosearch, Burlington, MA) for performing the solid phase sequencing and Bridget A. Clancy-Tenan for her expert typing of the paper. The many helpful discussions with Drs. Craig Gerard (Dept. of Medicine, Beth Israel Hospital, Boston, MA) and F. S. Heinemann (Dept. of Pathology, Hoag Memorial Hospital Presbyterian, Newport Beach, CA) are greatly acknowledged.

REFERENCES 1. Ziegler, D. M. (1988) in Drug Metabolism Reviews (DiCarlo, F. J., Ed.), Vol. 19, pp. l-32, Dekker, New York. 2. Ozols, J. (1989) Rio&em. Biophys. Res. Commun. 163,49-55. 3. Ozols, J. (1990) J. BioZ. Chem. 265, 10,289-10,299. 4. Gasser, R., Tynes, R. E., Lawton, M. P., Korsmeyer, K. K., Ziegler, D. M., and Philpot, R. M. (1990) Biochemistry 29, 119-124. 5. Lawton, M. P., Gasser, R., Tynes, R. E., and Philpot, R. M. (1990) J. Biol. Chem. 265, 5855-5861. 6. Yamada, H., Yuno, K., Oguri, K., and Yoshimura, H. (1990) Arch. Biochem. Biophys. 280,305-312. 7. Heinemann, F. S., and Ozols, J. (1984) J. Biol. Chem. 259, 797804. 8. Ozols, J., and Gerard, C. (1977) J. Biol. Chem. 252, 5986-5989. 9. Ozols, J. (1989) J. Biol. Chm. 264, 12,533-12,545. 10. Ozols, J. (1989) Biochim. Biophys. Actu 997, 121-130. 11. Hlavica, P., Kellermann, J., Henschen, A., Mann, K.-H., and KunzelMulas, U. (1990) Biol. Chem. Hoppe-Seyler 371.521-526. 12. Williams, D. E., Ziegler, D. M., Hordin, D. J., Hale, S. E., and Masters, B. S. S. (1984) Biochem. Biophys. Res. Commun. 125, 116122. 13. Williams, D. E., Hale, S. E., Muerhoff, A. S., and Masters, B. S. S. (1984) Mol. Pharmucol. 28,381-390. 14. Tynes, R. E., and Philpot, R. M. (1987) Mol. Phurmacol. 31, 569574. 15. Ozols, J. (1990) in Methods in Enzymology (Deutscher, M. P., Ed.), Vol. 182, pp. 225-235, Academic Press, San Diego. 16. Ozols, J. (1990) in Methods in Enzymology (Deutscher, M. P., Ed.), Vol. 182, pp. 587-601, Academic Press, San Diego. 17. Ozols, J., Heinemann, F. S., and Gerard, C. (1980) in Methods in Peptide and Protein Sequence Analysis, (Birr, C., Ed.), pp. 417429, Elsevier/North-Holland, Amsterdam. 18. Guan, S., Falick, A. M., and Cashman, J. R. (1990) Bin&m. Biophys. Res. Commun. 170,937-943. 19. Matsudara, P. (1987) J. Biol. Chem. 262, 10,034-10,038. 20. Jornvall, H. (1975) J. Theor. Biol. 55, l-12. 21. Nagata, T., Williams, D. E., and Ziegler, D. M. (1990) Chem. Res. Toxicol. 3, 372-376.

MINIPRINT

SUPPLEMENTAL

MATERIAL

FLAVIN-CONTAINING

SUPPLEMENT

TO MULTIPLE FORMS OF LIVER MICROSOMAL MONOOXYGENASES: COMPLETE COVALENT STRUCI-URE OF FORM 2.

by

Juis Ozols m - Peptides were attached to aryl amine membranes (Sequelon- AA; MilliCenlBiosearch, Division of Millipore Corporation), using procedures supplied by the manufacturer. The peptide was dissolved in neat TFA and applied to an aryl amine disk placed on a sheet of Mylar resting on a heating block at 550. After drying, the disk was wetted wth 5 pl of a 0.1 M solution of MES (2.N-morpholino)ethanesulfonic acid, containing 15% acetonitrile. and 10 mg/ml of EDC (1.ethyl-313. dimethylaminopropyl])carbodimide, pH 5.0. MES and EDC were obtained from Sigma. The coupling reaction was allowed to proceed at room temperature until the disk was dry (about 20 min). Membrane containing the coupled peptide was transferred to the reaction chamber of a Milligen/Biosearch 6600 ProSequencer sequencing system. Automated Edman degradations were performed using the standard 36 min protocol developed for this system by the manufacturer. FIX-amino acid derivatives were identified using an on-line HPLC system consisting of a MilliGen/Biosearch low dispersion pump, a SequeTag PTH analysis column (30 x 0.39 cm), and a Waters 490 multi-wavelength UV-VIS detector. F’TH amino acids were identified bv their characteristic elution positions and their UV absorption at 269 nm. The dehydro de&atives of wine and thnx&e were identified by their absorption at 313 nm. Data were collected and analwed usine the MilliGen/Biosearch Pm-Maxima chromatography package. Chromato~ams we& optimally aligned, using alignment algorithms. and then submxted to give a difference trace for successive sequencer cycles.

MINUTES Fig. S-2. HPLC of fraction 55. from column described in Rg. S-l, on a (I5 x 0.4 cm) C4 column. Solvent A was 0.1% TFA and Solvent B was 0.1% TFA in 75% acetonimle. A linear gradient from 0% to 100% of Solvent B in 70 min was performed at a flow rate of 1.0 ml/min.

Reduction. Carboxvami~ Large scale (IO nmol) chemical modifications of form 2 protein were performed as described in (3). Reduction and alkylation of peptides on a microscale level was performed by adding to the dry peptide the following solutions: 60 ~1 of 8 M urea, 10 ~1 of 2 M ammonium bicarbonate and 5 ~1 of 50 mM DlT. After 15 min incubation at 58, the mixture was cooled to room temperature, and 5 ~1 of 100 mM iodoacetamide was added. After 15 min, at room temperature, the mixture was lyophylized, dissolved in 88% formic acid and subjected to HFLC. -tic Cleavw Tryptic, chymoayptic and S. m protease digestions were carried out with I:25 w/w of the appropriate enzyme in 2 M urea. containing 50 mM ammomum bicarbonate, pH 8. The digestions were performed at room temperature for 18 h. Peptide cleavage with Asp-N protease was performed in 2 M urea containing 50 mM sodium phosphate buffer, pH 8.0, for I8 h at 370. Prior to the enzymatic cleavages protein or peptides were dissolved in 8 M urea. The urea concentration was then reduced to 2 M by the simultaneous addition of the appropriate buffer and the enzyme.

MINUTES Fie. S-3. HPLC of fraction 48 from column described in Fig. S-l. pe>ormed as described in Fig. S-2.

The HPLC was

Pvroelutamvl Aminooeutidase Treatment of Peotide R-16 -Two mg of lyophylized enzyme (0.5 units/80 mg of lyophylized material) was dissolved in 200 pl of IO0 mM sodium phosphate, pH 8.0, containing 5% glycerol, 10 mM EDTA and 0.5 mM DTT. At 50 pl aliquot of this solution was added to 1 nmol of lyophilized peptide. The tube was flushed wth N2 and incubated at room temperature for some I6 h. The mixture was dried, dissolved in 88% formic acid and subjected to HPLC. Chemicw For cleavage at methionyl residues, protein or peptide was dissolved in 70% formic acid to give about a 1% (w/v) protein solution, and a lO@fold molar excess of CNBr over methionine residues. After 18 h at room temperature, the mixture was partly dried under nitrogen in a fume hood, diluted IO-fold with water and lyophylized. For cleavage at methionyl and tryptophanyl residues, the dried peptide was dissolved in 0.2 ml each of neat heptafluorobutyric acid and 88% formic acid. After addition of about 250 mg of solid CNBr, the mixture was incubated at mom temperature for 18 h (8). The reagent and solvent were removed with a stream of nitrogen (in hood), diluted IO-fold with water and lyophylized. Cleavage at Asp-Pro bonds was performed by incubating the peptide with 88% formic acid at 370 for 72 h.

10

20

30

40

50

MINUTES Fig. S-4. HPLC of fraction 68 from column described in Fig. S-1. The HPLC was performed as described in Fig. S-2.

Electroblotting and sequence analysis of the blotted proteins were performed as described by Matsudara (19). Partially cleaved form 2 preparations were identified in the effluent fractions of HA-Agamse column upon elution with 300 mM KPi containing 0.1% NP-40. 20% glycerol, I mM EDTA and 0.1 mM D’lT (2.14).

FRACTION (6 ml/tube) I,lg. S-l. Gel filtration of tryptic digest of 6 nmol of carboxyamidomethylated and succinylated form 2 protein. The digest was applied to a column of LHa60 Sephadex (2.5 x IO0 cm) equilibrated wth formic acid/ethanol (3:7). Six ml fractions were collected at a flow rate of 10 ml/hour. The solid bar Indicates the distribution of a particular peptide in the fractions collected.

to

20

30

40

50

60

MINUTES Fig. S-5. HPLC of fraction 56 from column described in Fig. S-I performed as described in Fig. S-2.

The HPLC was

109

110

JURIS

IO

20

30

40

50

60

70 10

MINUTES Fig. S-6. Isolation of Peptide R-13 from fraction 40 of gel filtration column described in Fig. S-l.

FRACTION

OZOLS

20

30 40 MINUTES

50

60

Fig. S-10. HPLC of fraction 17 of Fig. S-7 using HPLC conditions as described in Fig s-2.

( 3 ml /tube)

Fig. S-7. Gel filtration of CNBr digest of carboxyamidomethylated form 2 protein. Approximately 4 moo1 of the protein was digested and applied to a column of LH60 Sephadex (1.5 x 95 cm) equilibrated with formic acid/ethanol (3:7). Three ml fractions were collected at a flow rate of 10 ml/hour. The dot indicates the kaction from which the indicated peptide was isolated.

IO

20

30

40

MINUTES kg. S-l 1. HPLC of fraction 32 of Fig. S-7 using conditions as described in Fig. S-2.

1

-I

IO

20

30

40

50

60

MINUTES Fig. S-8. HPLC of fraction 25 of Fig. S-7 using HPLC conditions as described in hg. S2.

MINUTES Fig. S-12. HPLC of fraction 29 of Fig. S-7. HPLC conditions are described in Fig. S-2

MINUTES Fig. S-9. HPLC of fraction 20 of Fig. S-7 using HPLC conditions as described m Fig. S2.

MINUTES

Fig. S-13. HPLC of fraction 22 of Fig. S-7 using HPLC conditions as in Fig. S-2

COVALENT

STRUCTURE

OF LIVER

MICROSOMAL

MONOOXYGENASE

FORM

I

111

2

9-10 R-IO. E-2

-0

R 2

\

:;.i IO

20

30

40

MINUTES MINUTES I,lg S-14. HPLC resolution ~~mdlt~ons as in Fig. S-2.

I 0 0 M a

of Asp-N

protease

digest

CB-4

and 5 u>lilg

20

30

I

40

MINUTES

1.1g. S-l.5 Srsnhvlwxcus

Isolation w

of PeptIde CB-9,10;E-6. protease and the digest

IO

20

I,lg. S-18. Isolation of Peptide R-lO;E-2. m protease. The dtgest was resolved

The cleavage was performed by HPLC as in Fig. S-2.

wth

Stanhvloccu\

I 1, O c a

iI! 10

of Peptides

The parent peptlde resolvd as 1” Fig S-2

30

40

was digested

~1111

I

! 10

20

---l-

30

40

50

MINUTES

F!g S-19. Isolation of Peptide R-lZ;D-8. 20 of Fig. S-l was digested with Asp-N F1g. s-2.

Partially purified PeptIde R-12, from fraction protease and the digest resolved by HPLC ii\ m

50

MINUTES 10

I-lg. S 16. Cleavage of Asp-Pro bond in Peptide CB-14. The peptlde (m 88% formic aad) was incubated for 72 h at 370. The HPLC of the digest was perfomxd as in Fig So 2

Fig. S-20. HPLC of $taDhvlowaxs digest was pefomwd as in Fig. S-2.

I IO

20

30

40

MINUTES I+g. S-17. HPLC rsolation of Peptides CB-14;W-4 and CB-14;W-5. Cleavage of l’r[~ re\ldues in CB-I4 was performed by CNBr as described under “Expertmental Sectmn” HI’LC of the digest was performed as in Fig. S-2.

20

30

40

MINUTES

15

m

digest

20 FRACTION

of Peptlde

25 (3

R-l?.

30

The resolution

ot tk

35

ml/tube)

kg. S-21. Gel filtration of tryptlc digest of form 2 protein. About three nmol of d!gewd protein was carboxymethylated and the reaction mixture applied to n column of LHhO Sephadex (I .5 x 95 cm) equihbrated with formic acid/ethanol (3:7) Three ml fractmn\ were collected at a flow rate of 10 ml/hour.

112

JURIS

10

20 30 MINUTES

OZOLS

40

Fig. S-22. HPLC of fraction 24 of Ftg. S-21 using conditions as m Fig. S-2.

-

Fig. S-24. HPLC of fraction 27 of Fig. S-21 using conditions as in Fig. S-2.

02

I

P 4” 01

4

IO

I 20

r

I 30

40

h IO

I

I

1

1

1

II

20

30

40

50

60

70

MINUTES

MINUTES

Fig. S-23. HPLC of fraction 30 of Fig. S-21. Peptlde R-16 denotes nyptic peptide, residues 486 tc~494.

Tsblp DOS. 19 ii' 21 22 23 24 25 2G 27 2s 29 3c 31 32 33 34 3.5 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

SI.

Sequence Data for Few 2 llonoxyqenase Peptidfa [cycle), yield (pmol)

seq. Ser cvs Lk" GlU Glu Gly LeU Glu Pt-0 Thr CVS Pie Glu "let Ser ,\sp As17 Ile Cl; Gly LelJ Tr?t LYS Phe Ser PSll 'iii Pla SlU Gl" Gly h-g Pla ser !le T‘/ r

POS. R-2 (1) (2) (3) (4) (5) (6)

4:, 22 ?J 25 27 16

17; P (9) (10) ill! (12) (131 (14) (15) (16) (171

:: 11 5.7 6.5 7.6 7.1 4.3 1.9 3.2 3.5

R-3 (1) (2) (3) (4)

9.1 3.5 7.9 8.0

Fig. S-25. HPLC of fraction 22 of Fig. S-21 using conditions as in Fig. S-2.

& (4)

(9) (101 (11) (12) (13) (14) (15) (lh) (17) (131 (19) (20) (21)

;; li

G.2 7.0 3.1 6.2 2.4 4.9 4.0 4.2 3.7 2.0 2.7 1.6 2.3

seq.

55 36 57 3& 59

Gin Ser Val Phe Thr

63 64 65

LYS Glu Islet

71 72 73

78 7" x0 ill 42 83 84 85 X6
Peptides

(5)

7.3

I:] (8) (9) (10) (11) (12)

::," 4.9 3.1 1.G 0.3 0.4

I;;] (15) (16)

;:6' o.:, 1.1

T-6 (1) (2)

Phe Pro Pi?e

Pro Asn Asn Net Vi * iisn Ser Lys Leu 61n Gil! Tyr Ile Thr

m-4 (1)

34

1:;": 1 [g;;; (hj 79 (7)10! (~)!20

(9) 97 (10)

23

?II 38

ii] (5)

(6)

",: 31 2'

iii (9)

5: 19

ii:; (12) (13; (l&j

:3 14 '1 9.8

liS', (17) (18) (19)

::4 3.4 2.3 0.5

i;:;

A::

COVALENT

STRUCTURE Table

SI.

OF LIVER

MICROSOMAL

Sequence Data for Form 2 ~lononyqenase Peptidea (cycle), yield (pmol)

PUS.

Seq.

Pas.

seq.

92 13 94 95 36 97 33 39 1X 101 102 103 104 10s 136 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 12R 129 130 131 132 133 134 135 136 137 131! 139 140 141 142 143 144 145 146 147 148 143 150 151 152 153 154 155 156 157 158 159 160 161 162 lb3 16U 165 166 167 168 169 170 171 172 173 174 175 176

Thr Phe Ala An3 Glu LYS Asn LeU LfZU LYS TY~ Ile Gin Phe Lys Thr Leu '!a1 Ser Ser Ile LYS LYS tlis Pro ASP Phe Ser Val Thr G'Y Gin T!"D TY~ Val Ala Thr W Arq ASl. Gly iys LVS Glu Thr Ala va1 Phe ASD Al; Vdl Met

177 178 179 180 181 182 1 X?

GlU Pt-0 Gl .v Ile Phe i.ys Glv

184 185 ! 86 167 188 1x9 190 191 192 193 134 195 196 !Y7 198 199 LUti 2Lil 202 203 204 205 206 2d7 200 209 210 211 212 213 214 215 216 217 '1X 219 220 221 222 223 224 225 226 227 223 229 230 231 232 233 234 235 236 737 233 239 240 241 243 242

LYS AV 'Val Leu Vai Ile Gly LW GUY Asn Ser GUY Gl u

R-4 (1) (2) (3) (4) (5)

21 24 252 38 42

1;; (3) (9) (10) (11) ;;;]

;i 26 20 24 22 ;;

(14) (15) (16) (17) I;;{

20 11 13 12 ;;

(20) (21) ;;;I

6.2 4.3 33::

(24)

1.2

CS-4,D-2

i:i E Iii k 12;::i

(7) (3) (9) (10) i;:i (13) (14) (15) (16) (17)

R-5 i:j

:;

(7) (8) (9) (101 illj (12) (13)

33

5.5 1.1 4.3 4.8

::; 3.2 0.2 1.5 0.8 0.2

24

30 25 24 21 2u

I’e cys

1;;; ;;

Ser GUY ris HlS Val Tvr Pro ASn LeU Pl-0 LYS ASP Ser Phe Pro Gly LeU LYS His Phe LYS GUY LYS Ser Phe ArrJ Gln At-3 GlU TY~ LYS

(16)

7.7

,D-2 :: 29 21 17 15 11 3.0 8.7 6.9 6.3 5.8 1.9 4.5 1.5 2.1 0.6

R-6 (1)

33

(3)

25

(2) 23 CB-5,D-3

MONOOXYGENASE

244 245 246 247 248 249 250 25' 252 253 254 255 256 257 258

261

FORM

2

Peptides

(4) (5) (6) (7)

28 17 21 13

R-7

(4)

23

(7)

15

(10) (11) (12)

14 12 10

tzii: i:i1y

ASP

Ile 01 a Thr Glu Leu Ser

Hi 5 Thr Ala Glu I;ll. Val Vzl Ile

CB-5,D-7 iii 143; (5)

i:: 7.9

(6j 2.5 iii 2

Ser

SfZr Arg SPIGly SetTOP Val Met ier Arq va1 TOP AS0 ATI1 tily TY~ PI-o Trp Asp llet LelJ TY~ Val TtIr Arii Phe Gltl Thr Phe LW Lys ASl. ASll Leu Pl.0 Thr Ala Ilf Ser ASP Trn Tt-0 TY~ Val LYS Gln Het ASn Ala

;o"

T-l& (1) (2) (3)

2.5 7.0 1.5

(9) (10) (11)

0.3 5.7 3.3

R-9

(1) 15 i:i 2;

(4)

!5i

7.3 9.3

:;l 2 (8) 3.75.8

CB-7 (1) (2)

R-10 (11137 (2)11@

J'J 17

iii 7:

(5) (6) (7)

10 18 17

(8) 10 i;",'lZ 151 ,~I 89 ~~ i:i (3) (9) 10) 11) 12) 13) 14) !5) 16) I71 18) 19) !O) !l) !2) !3) '4)

:: 63 45 38 23 18 16 5.0 14 7.1 7.0 13 11 5.9 6.3 9.4 6.1 3.3

CR-8

(9) (10) ill) (12)

7.3 6.5 5.7

113

114

JURIS Table

SI.

Sequence

Data

Peptidea POS.

seq.

ZuL 263 264 265 266 267 263 269 270 271 272 273 274 275 276 277 278 279 280 231 282 283 284 205 286 287 283 239 290 291 292 293 294 295 296 297 290 239 300 301 302 303 304 305 306 307 303 309 310

LyS Phe LYS Hi 5 GlU Asn TY~ Ser LIX, Plet Pro Leu Asn Gly Thr Leu Arg LYS Glc PI-0 Val Pile ASll PSp 4sp LW pro Ala RI-Cl Ile Leu CYS GUY Thr Val SetIle LYS Pro PSll Val LYS Glu Phe LYS GlU Phe Thr Gl i,

311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345

Thr SetAla Ile Phe GlU ASP Gly Thr Val Phe Glu Ala Ile ASP Ser Val Ile Ph!? Ala Thr Gly TY~ Gly TY~ Ala TY~ Pro Phe Leu ASP ASP ser

(25)

12.0

R-lO,E-2 (1) 38 (21 47

(23)

R-lZ,D-8 I:] :: (3)

14

1%

8.7 8.2

1;;;

2:

i;ti

i:;

I;:] (12) (33)

4":: 3.7 3.1

1:;; (36) (37)

i:: 1.0 0.2

!;;I ;:;: I;;\

;:;

35 i;:j (23)

::: 3.7

T-27 (1) (2) (3) (4)

53 65 49 45

(5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19)

40 20 43 37 38 28 26 31 20 28 25 17 20 18 11

OZOLS

for Fov 2 llonoxyqenase (cycle), yield (pmol) POS.

Seq.

34G 347 34d 349 350 351 352 353 354 355 356 357 358 359

LYS Ser GlU Asn Asn Lys 'ial Thr LeU Phe LYS GUY Ile Phe

361 362 363 364 365 36b 367 368 369 370 371 372 373 374 375 376 377 378 379 330 381 382 383 3x4 385 386 3x7 383 33Y 390 391 392 393 394

Pt-0 Gln LeU GlU Lys PI-0 Thr Yet Ala Val Ile GUY LW Val Gln Ser Let. Gly Ala Ala Ile Pro Thr Thr ASP L?U Cln Ala Arg Trp Ala Ala Gln Val

3% 3% 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 4211 429

I'e LYS GUY Thr CYS Thr $

(6)

55

1;; (9) 110)

:," 55 60

(24) (25) (26)

1.1 0.4 0.6

CD-11

(9)

40

ii:; (12)

:i' 52

Pro Val LYS ;%&

I;:] (15)

:: 18+llet

j;;i

;;

;:A

I;:] (20) (21) (22)

;: 10 7.2 4.2

ASP Il!? His Glu Lys Met GUY Thr LYS LeU LYS Thr Phe Gl Y iy; Tl-D Gl; Thr Ile Gln

Peptides

R-13;E-2 i:]l! 13) (4) (5)

3

CB-12 (1) 3l

(2) 20

(3) (4) (5)

19 8.6 15

90 51 50 (5) 38 161 30

(10)

12

'(;:j (13) (14)

:: 30 23

R-13;E-3

COVALENT

430

Thr

(4)

431 432 433 A34 4?5 436 437 43:: 33Y 440 ‘44 1 442 443 444 445 445 447 43c 449 450 451 45; 453 454 455 45b 457 455 439 460 461 462 463 464 465 466 467 468 465 470 471 372 473 474 475 47h 471 473 479 480 481

Asp

(5)

Tyr IliZ

Rsn i7r :.1et

Ih) (71 (8) (9) (10)

Asr1 Cl!) GL (12) Lelc &la SetFhe :1e Gly !,a1 LYS I eu 4sn Ile PI-0 :rp LEU Phe Lt?lJ :hr As ,I PI"0 ill-9 I.CU Ala Lfll Gl li l'al Dhe Php Gly PTO ryi ser Pm lYr Clll Uhe Arq LCU Val Gly fro Gly L '/ s Tl-1'

(13j

STRUCTURE

OF LIVER

MONOOXYGENASE

FORM

2

115

50 6X 60 63 51 45 38 23 17 13

CR-14 (I)117 (21124 (3)11X (4j136

I:] ii (7) 84 (8) 78 (9) (10)

92 57

1;;; (13) (14)

63 sl 68 56

1-l;;

41;

(17) (If) (19)

41 37 10

;;y; (22) (23)

:;

1;@P-2 71 63 127 I3Y 125 95 95 102 I26 95 42 40 16 32 26 23 24 13 21 15 11 7

7.1 10.0

‘1-15 (11101 (2) 84 (3) 57

'In all succinylated methvlCystelne or

MICROSOMAL

peptides Lys carboxvmethylcvstcine.

Table

Residue

Position 19 9% 102 115 125 278 339 405 454 456

1s identified

487 483 GSY 490 411 492 493 444 495 496 497 4YC 499 500 501 502 x3 504 505 506 5d7 sub 509 510 511 512 513 514 515 516 517 51 ;I 519 520 ii1 522 522 124 525 5Zf, 527 523 x:li 533 531 i>3: 533

ds 5%Lvs;

SII. Di:ferences llonooxyoenase (3)

Between and the

Al a 1le LCU Thr Gill -rp !Ai[! ,4rq set. LfU I.YS PK ?1et ! ys TiltAt-q illa va1 Cl L, 111s Leu 61 II LVS PK ila LelJ Phe SL'r Pro GlU Leu Tl-1' LElJ LelJ Al a Ile I1 a ':a 1 Lfll LfU !le A! a ii 1 3 ildl LEU \'?I1 Phe irl

slkylated

i4) /5i

16 14

(6: 4 (7) 7 (JJ 2

:!!-5 is 16 11 l? :1 7

k-17 ilj34 (2) 41 (3) 32

R-l:i('ol.Pharei (1: 2 !2! 7

(6) !7! (Cl 19) (10) (11) (12) (13) (141

25 17 23 12 2.4 4.1 4.3 6.0 3.5

peptides,

the Prlnlar‘y Structure cDNP Encoded Seq~~cnc~ PTII (3)

CD-14 (1) (2) !3! ,4: j5i (6)

I!!

::

iojj (7) !:i)

;7 IL1 2.6

--4L (1 !X!l :2;162 !3)100 (4) 31 (5) 6:’ (6) 41 (7) 15 (51 19 (9) 17 Ii? (121 (13) (14) (151

Cys

1s identified

of For'!? 1 (5)

rls cdrboxyarnrlo-

lo'3 4 ?.'I 3.1 l.i, 0 5