supporting information - wiley-vch · yang et al supporting information page 2 k a and k b are the...

Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2005

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2005

Supporting Information

for

High-Throughput Mass-Spectrometry Monitoring for Multisubstrate Enzymes: Determining the Kinetic Parameters and Catalytic Activities of

Known and Novel Glycosyltransferases

Min Yang, Melissa Brazier, Robert Edwards, and Benjamin G. Davis*

More Detailed Kinetic Analysis

For two substrate reactions as catalysed by GTs, steady state assumptions may be

reliably applied to multi-substrate enzyme reactions.[1] King-Altman analysis [2] allows

the rate equation of an "Ordered Bi Bi" reaction, using the nomenclature of Cleland [3]

(Scheme 1) to be written as follows:[4]

k1k2k3k4[E0] − k-1k-2k-3k-4[E0] υ = ------------------------------------------------------------------------------- {1}

k-1k4(k2 + k3) + k1k4(k-2 + k3)[A] + k2k3k4[B] + k1k-2k-3[A][P] + k-3k-4(k-1 + k-2)[P][Q] + k2k-3k-4[B][P][Q] + k1k2k-3[A][B][P] + k1k2(k3 + k4)[A][B] + k2k3k-4[B][Q] + k-1k-2k-3[P] + (k3 + k-2)[Q]

For initial-rate studies such as those described here, this may be simplified with the assumption [P] = [Q] = 0 to give:

Vmax[A][B] υ = ---------------------------------------- {2}

KIAKB + KB[A] + KA[B] + [A][B]

Where Vmax = k3k4[E0] / (k3 + k4) KA = k3k4/(k1(k3 + k)) KB = k4(k-2 + k3) / (k2(k3 + k4)) KIA = k-1/k1

Yang et al Supporting Information page 2

KA and KB are the KMs for each substrate and KIA is the dissociation constant for

the (EA) complex. With a fixed concentration of one substrate, e.g. A, a

Lineweaver-Burk plot analysis, e.g. 1/ vs. 1/[B], can be performed.

]A[V

]A[K

]B[

1)]A[

K1(

V

K/1

max

AIA

max

B +++= {3}

It is clear from {3} that if the concentrations of A are in great excess of KIA, the

variation in slopes of 1/ vs 1/[B] at different [A]s are poorly detectable. To avoid

this source of imprecision, the concentrations of both substrates in the current

experiments were kept low (20 -100 µM).

For Random Bi Bi reactions (Scheme 2),King-Altmann analysis coupled with

computational assessment[5] allows the following to be derived.

22

ih

2

g

2

fedc

22

ba

]B][A[]B[]A[K]B][A[K]B[K]A[K]B[K]A[KK

]B][A[]B[]A[K]B][A[K(V

+++++++

++= {4}

The definitions of the various kinetic constants, Ka – Ki, differs from those in the

ordered mechanism.[6]

The Random Bi Bi kinetic equation {4} maybe simplified with the assumption that

all steps other than the central conversion of EAB EPQ are in rapid

equilibrium. The equation for this system (Rapid Equilibrium Random Bi Bi) can

be obtained from the more complex equation {4} by eliminating all the terms that

contain either of the rate constants for the central EAB-to-EPQ step. This is

typically a reasonable assumption, since the turnover stage is often the most

critical and therefore rate limiting step. Thus, in both circumstances, use of the

REA allowed the use of simplified equation {2}. The resulting rate equation is

then identical in form to the ordered Bi Bi but without terms in [A][P], [B][Q],


[A][B][P], and [B][P][Q] in the denominator. This conveniently leads to the same

general equation {2}. The similar form proves ideal for parallel data analysis

although it should be noted that the interpretations of some resulting constants

crucially differ. Definitions for the kinetic constants are the same except that KIB =

constant/coefB and KIP = constant/coefP.[1]

Methods for Bisubstrate Enzyme Mechanism Determination

To determine a given bisubstrate enzyme mechanism, two methods are typically

employed: product inhibition or dead end inhibition.[4, 7] Due to the availability of

products, product inhibition is the most commonly used while dead end inhibition

requires the use of two distinct inhibitors, one of the donor and another of the

acceptor. Although convenient, compared with dead end inhibition, product

inhibition is less informative, since only one competitive and yet 3 mixed type

inhibition patterns result from all the possible inhibitor-substrate variation

permutations of product inhibition of an Ordered Bi Bi mechanism. In contrast,

through the use of dead end inhibition, 3 different types of inhibition pattern can

be expected and determination is thus simplified. Yet due to the lack of

availability of suitable dead end inhibitors, usage of this approach is restricted.


Lineweaver-Burk Analyses

Additional Figure 1 Lineweaver-Burk plot of -GalT. Characteristic intersection

in the 3rd quadrant indicates that KIA < KA.

-40000

20000

80000

-0.06 -0.04 -0.02 0 0.02 0.04 0.06

100 µM

20 µM

60 µM

1/ /minámM-1

1/[UDPGal] /µM-1

Additional Figure 2 Kinetic results for UGT72B. 1/ vs. 1/[UDPGlc] plotted at

different [DCA] (Figure 7) yielding a plot with intersection in the 2nd quadrant

characteristic of KIA > KA. Kinetic parameters were then obtained through non-

linear regression.

0

70000

140000

-0.015 -0.005 0.005 0.015 0.025 0.035 0.045 0.055

1/[UDPGlc] /µM-1

1/ /minámM-1

100 µM

20 µM

60 µM


Comparative Survey and Correlation of KA/KB for Prior Bi-Bi Kinetic

Analyses

(a) Ordered Bi Bi mechanism:

Macrolide glycosyltransferase (OLED)

KB (UDPGlc)/KA (Lankamycin) = 599.6 µM / 33.17 µM =18.07[8]

Macrolide glycosyltransferase

KB (UDPGlc)/KA (Oleandomycin) = 21.57 µM / 2.9 µM = 7.43[9]

Heptosyltransferase I

KB (ADP-Mannose)/KA (Kdo2-lipidIVA) = 1.5 mM / 4.5 µM = 333[10]

Lipase Novozym 435

KIB (Ac)/KIA (Al) = 0.0653 M / 0.0022 M = 29.68[11]

NAD(P)-dependent glucose 1-dehydrogenase (GDH)

KB (NADP)/KA (Glucose) = 11.3; KB (NAD)/KA (Glucose) = 9.1[12]

Novozym 435

KB (H2O2)/KA (Lauric acid) = 11.5 [13]

sn-glycerol 1-phosphate dehydrogenase

KB (dihydroxyacetone phosphate)/KA (NADH) = 0.46 mM / 0.032 mM =14.38 [14]

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate

synthase (HPPK/DHPS)

KB (ATP)/KA (Dihydropterin) = 70 µM/1 µM = 70.00 (pea); KB (ATP)/KA (Dihydropterin) = 70

µM/2 µM = 35.00 (recombinant) [15]

Thio methyltransferase Isoform II


KB (AdoMet)/KA (I-) = 34 _ 103 /70 = 485; Isoform III KB (AdoMet)/KA (I

-) = 2.3 _ 103 /52 =

44.23; Isoform V KB (AdoMet)/KA (I-) = 0.37 _ 103 /21 = 17.61 [16]

(b) Random Bi Bi:

Cellobiose phosphorylase

KB (Glucose)/KA (Glucose 1-phosphate) = 0.29 mM/0.15 mM = 1.93[17]

Adenylate kinase

KB (AMP)/KA (MgATP) = 0.12 mM / 0.06 mM = 2.00[18]

NADPH-cytochrome P450 oxidoreductase (CPR)

KB (cyt c)/KA (NADPH) = 1.59 µM / 1.46 µM = 1.09 (Native); KB (cyt c)/KA (NADPH) = 1.12

µM / 1.41 µM = 0.79 (Soluble)[19]

Rapid equilibrium random Bi Bi:

p21-activated kinase (PAKs)

KB/KA = 71µM / 17 µM = 4.17 (KIB/KIA =147 / 35 = 4.20) [20]

mdm2

KB (og-Ub Ubc4)/KA (p53) = 3.2 µM/ 1.0 µM = 3.2[21]

Polynucleotide phosphorylase

KB (Pi)/KA (polyA) = 0.62 mM / 83 µM = 7.47[22]


Representative TIC Timecourse Plots

Additional Figure 3 -GalT product TIC time course monitored by MS. Peaks

show TIC for the generation of product (galactosyled MUGlcNAc) as a function of

time at [UDPGal] = 60 µM and [MUGlcNAc] = 100 µM.

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25

Time0

100

%

2.00

1.89

1.72

1.090.970.310.15

2.03 2.17 2.35

2.47

2.61 2.71

2.862.99

3.163.31

4.07 4.21 4.94

t = 180 min

t = 147 min

t = 113 min

t = 80 min

t = 47 min

t = 13 min

t = 0 min

From the bottom to the top: t = 0, 13, 47, 80, 113, 147, 180 min

Additional Figure 4 UGT72B1 product TIC time course monitored by MS. Peaks

indicate the generation of product (glycosylated 3,4DCA) catalyzed by UGT72B1

as a function of time (t = 0 at the bottom) at [UDPGlc] = 60 µM and [3,4DCA] =

100 µM.

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25

Time0

100

%

t = 181 min

t = 147 min

t = 114 min

t = 80 min

t = 47 min

t = 14 min

t = 0 min

From the bottom to the top, t = 0, 14, 47, 80, 114, 147, 181 min


[1] S. Ainsworth, Steady-State Enzyme Kinetics, the MacMillan Press LTD,London, 1977.

[2] E. L. King, C. Altman, J. Phys. Chem. 1956, 60, 1375.[3] W. W. Cleland, Biochim Biophys Acta FIELD Publication Date:1963 Jan 8

1963, 67, 104.[4] D. V. Roberts, Enzyme kinetics, Cambridge University Press, New York,

1977.[5] R. O. Hurst, Canadian Journal of biochemistry 1969, 47, 941.[6] M. Dixon, the Enzymes, LongMans Green and Co Ltd., New York, 1958.[7] A. Cornish-Bowden, Fundamentals of Enzyme Kinetics, Portland Press,

1995.[8] L. M. Quiros, R. J. Carbajo, A. F. Brana, J. A. Salas, the Journal of

Biological Chemistry 2000, 275, 11713.[9] L. M. Quiros, J. A. Salas, the Journal of Biological Chemistry 1995, 270,

18234.[10] J. L. Kadrmas, C. R. Raetz, JOURNAL OF BIOLOGICAL CHEMISTRY

1998, 273, 2799.[11] T. Garcia, N. Sanchez, M. Martinez, J. Aracil, Enzyme and Microbial

Technology 1999, 25, 584.[12] T. Ohshima, Y. Ito, H. Sakuraba, S. Goda, Y. Kawarabayasi, Journal of

Molecular Catalysis B: Enzymatic 2003, 23, 281.[13] G. D. Yadav, K. M. Devi, Biochemical Engineering Journal 2002, 10, 93.[14] J.-S. Han, Y. Kosugi, S. Ando, H. Ishida, K. Ishikawa, in Jpn. Kokai

Tokkyo Koho, (Sangyo Gijutsu Sogo Kenkyusho, Japan). Jp, 2002, p. 12pp.

[15] J.-M. Mouillon, S. Ravanel, R. Douce, F. Rebeille, Biochemical Journal2002, 363, 313.

[16] J. Attieh, S. A. Sparace, H. S. Saini, Archives of Biochemistry andBiophysics 2000, 380, 257.

[17] E. Rajashekhara, M. Kitaoka, Y.-K. Kim, K. Hayashi, Bioscience,Biotechnology, and Biochemistry 2002, 66, 2578.

[18] X. R. Sheng, X. Li, X. M. Pan, Journal of Biological Chemistry 1999, 274,22238.

[19] D. C. Lamb, A. G. S. Warrilow, K. Venkateswarlu, D. E. Kelly, S. L. Kelly,Biochemical and Biophysical Research Communications 2001, 286, 48.

[20] H. Wu, Y. Zheng, Z.-X. Wang, Biochemistry 2003, 42, 1129.[21] Z. Lai, K. V. Ferry, M. A. Diamond, K. E. Wee, Y. B. Kim, J. Ma, T. Yang,

P. A. Benfield, R. A. Copeland, K. R. Auger, JOURNAL OF BIOLOGICALCHEMISTRY 2001, 276, 31357.

[22] J. Y. Chou, M. F. Singer, P. McPhie, JOURNAL OF BIOLOGICALCHEMISTRY 1975, 250, 508.

72B1

UDPG

1 2 3 4 5 6 7 8 9 10 11 12

13 24

25 36

37 48

49 60

61 72

73 84

85 96

3. Umbelliferone [M+ Cl-]- 359

notebook 2 page 77

180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z0

100

%

qualification 4-3 116 (2.152) Cm (80:183) Scan ES- 4.50e3359

187

177

197

201214 323229 231 282241 269255 313283 303 349339329

361403

369386

383 387404

565425 461440 445 483477

523510500 541561

587566 606601 612

OHO OO

OH

O

HOHO

HO O O

OHOHO

OOH

OH

P OOH

OP OO-

O

O

OHOH

HNN

O

O

C15H23N2O17P2-

Exact Mass: 565.0477

C9H6O3Exact Mass: 162.0317 C15H16O8

Exact Mass: 324.0845

4. 4-methylumbelliferone [M + Cl-]- 373

C15H23N2O17P2-

Exact Mass: 565.05Mol. Wt.: 565.29

OHOHO

OOH

OH

P OOH

OP OO-

O

O

OHOH

HN

N

O

O

OHO O

CH3

C10H8O3Exact Mass: 176.05

Mol. Wt.: 176.17

O

OH

O

HOHO

HO O O

CH3


Mol. Wt.: 338.31

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z0

100

%

qualification 4-4 122 (2.262) Cm (115:129) Scan ES- 943403.13

282.20

241.18

272.16243.14

351.22

313.27

283.05

286.11

315.25

323.17 335.15

373.20359.19

375.16401.40

393.28

565.15

404.14425.13 508.22482.95461.06453.22

496.40 525.10 550.76

587.15

566.26

567.35 588.29

8. 3,4-dichloroaniline, [M + Cl-]- 358

notebook 2 page 77

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z0

100

%


210

198

199

226

211

212 245229 247 256 267 349315281

313290 339323

360

362403385

368 370

387389 404

425420 521429509437 477

448464 506

484 565539 550 566 600589607

O

OH

HOHO

HO

NH2

Cl

Cl

NH

Cl

Cl

C6H5Cl2NExact Mass: 160.98

Mol. Wt.: 162.02

C12H15Cl2NO5Exact Mass: 323.03

Mol. Wt.: 324.16

C15H23N2O17P2-


OHOHO

OOH

OH

P OOH

OP OO-

O

O

OHOH

HN

N

O

O

9. 3,4-dihydroxyl benzoic acid [M-H+]- 315.08

notebook 2 page 77

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z0

100

%


199

209

226

211

213 358315245230 263247 265 292281 313 351323

329349

360 403362 385 387 565

404 507430425

506493

440 476449457463 558

510525 547 587

571596 597 606

O

OH

HOHO

HO

COOH

OHOH

O

COOHHO

C13H16O9Exact Mass: 316.08Mol. Wt.: 316.26


Mol. Wt.: 154.12

C15H23N2O17P2-


OHO

HO

O

OH

OH

PO

OH

O

PO

O-

O

O

OHOH

HN

N

O

O

10. 2,5-dihydroxyl benzoic acid [M-H+]- 315 [M+Cl-]- 351

notebook 2 page 77

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z6

100

%

qualification 4-10 131 (2.427) Cm (75:175) Scan ES- 953323

226

315

265259228245

241 247

252

313267274 311290

362

351349329335

403

381373 383

404

433431409 435 545508443 461 471 486491 510 536

531 604587559565

584 611

C15H23N2O17P2-


OHOHO

OOH

OH

P OOH

OP OO-

O

O

OHOH

HNN

O

OO

OH

OHHO

HO

COOHOHHO

O

HOOC



HO

30. 7-hydroxy 6-methoxy coumarin (Scopoletin) [M+Cl-]- 389

O O

H3CO

HO


Mol. Wt.: 192.17

O O

H3CO

OOHO

HOOH

OH


C15H23N2O17P2-


OHOHO

OOH

OH

P OOH

OP OO-

OO

OHOH

HNN

O

O

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z0

100

%

qualification 4-30 104 (1.932) Cm (87:106) Scan ES- 2.56e3389.18

282.20

239.10 255.23248.27 265.22

323.15312.23283.06329.28

349.16382.43

403.17

565.19

404.03

416.20 425.13453.11 483.17

461.05522.23506.34 540.95 557.41

587.13566.20603.06

31. 6,7-dihydroxy coumarin (Esculetin) [M-H+]- 339)

O O

HO

HO


Mol. Wt.: 178.14

O O

HO

OOHO

HOOH

OH


Mol. Wt.: 340.28

C15H23N2O17P2-


OHOHO

OOH

OH

P OOH

OP OO-

OO

OHOH

HNN

O

O

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z0

100

%

qualification 4-31 96 (1.785) Cm (82:96) Scan ES- 4.71e3339.22

282.19

240.11 254.99265.16

323.21283.11312.19

403.15

353.17

340.22

354.21375.13

362.24402.19 565.15404.04

425.06 453.21 482.99461.08

515.22506.86 522.29544.99 587.18566.15

601.32

32. Amber resultDihydrojasmonic acid [M-H+]- 373, [M+Cl-]- 409

O

OH

O


O

O

O

OHOHO

OH

OH


C15H23N2O17P2-


OHOHO

OOH

OH

P OOH

OP OO-

OO

OHOH

HNN

O

O

280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z10

100

%

qualification 4-34 98 (1.822) Cm (97:102) Scan ES- 285565

349

313281

283

290 307295 296

335332

317 331

344

337

403351

373371

353

365357

381

383

389393

409436

425423429

507450

448 451 473464 489479

506501

508544538519 531 554

599567587577 606

3

24

36

48

60

72

84

96

4

8

9

10

30

31

O

OH

HO

OOH

OH

P OO-

OP OO-

O

O

OHOH

HN

N

O

O

NH

N

NO

NH2N

OHOHO

O

OHOH

P OO-

OP OO-

O

O

OHOH

SHOHO

O

OH

OH

PO

O-

OP

OO-

O

O

OHOH

HN

N

O

O

NH

N

NO

NH2N

OP OO-

OP OO-

O

O

OHOHO

OHHO

OH

OHO

HO

OAcHN

OH

P OO-

OP OO-

O

O

OHOH

HNN

O

O

OHOHO

O

OHOH

P OO-

OP OO-

O

O

OHOH

HNN

O

O

3. UDPMan + Umbelliferone [M-H+]- 323

notebook 2 page 97 72B1

180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580m/z0

100

%


241

177192

187 194231211201 227

403323259255 280262 282 311291

344325 342 363

360 364 383 385

522442404

425 453 491464485

465 493 511562523

531 557

587566

567 588

OHO OO

OH

O

OH

HOHO O O

C15H23N2O17P2-



Mol. Wt.: 162.14


Mol. Wt.: 324.28

OHO

HO

O

OHOH

P OOH

OP OO-

O

O

OHOH

HN

N

O

O

8. UDP5SGlc + 34DCA [M + Cl-]- 374)


330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590m/z0

100

%


374

338

329

340

360342358 362

403

378

380 401385

404425420

581490483426 448436 459458 467471 565563507491501 515 529528 543541 549 579 582587

SHO

HO

OOH

OH

P OOH

OP OO-

O

O

OHOH

HN

N

O

OS

OH

OH

HOHO

NH2

Cl

ClNH

Cl

Cl

C6H5Cl2NExact Mass: 160.98Mol. Wt.: 162.02

C12H15Cl2NO4SExact Mass: 339.01Mol. Wt.: 340.22

C15H23N2O16P2S-


8. UDPMan + 3,4DCA M 323, [M+Cl-]- 358, 360


300 320 340 360 380 400 420 440 460 480 500 520 540 560 580m/z0

100

%


442

362

323

311291297 299321

344

325339

360345351

403

363

364 383377 384 400 404425410 430

522490464443

453447 454 486480

465 491500506511 562

523544535 545

587566

567584579 588

598

NH2

Cl

Cl

NH

Cl

Cl

C6H5Cl2NExact Mass: 160.98Mol. Wt.: 162.02

C15H23N2O17P2-


OHO

HO

O

OHOH

PO

OH

OP

OO-

O

O

OHOH

HN

N

O

O OHO

HO

OHOH

C12H15Cl2NO5Exact Mass: 323.0327

Mol. Wt.: 324.1572

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