Udpglucose dehydrogenase Kinetics and their mechanistic implications

25

Biochimica et Biophysica Acta, 481 ( 1 9 7 7 ) 2 5 - - 3 2 © E l s e v i e r / N 0 r t h - H o l l a n d B i o m e d i c a l Press

BBA 68068

UDPGLUCOSE DEHYDROGENASE KINETICS AND T H E I R MECHANISTIC IMPLICATIONS *

A L F R E D B. O R D M A N a n d S A M U E L K I R K W O O D **

Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minn. 55108 (U.S.A.) ( R e c e i v e d S e p t e m b e r 1st, 1 9 7 6 )

Summary Initial velocity and product inhibition studies were carried out on UDPglucose dehydrogenase (UDPglucose: NAD* 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD ÷ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD ÷. Inhibition by NADH gave complex patterns. Lineweaver-Burk plots of 1/v versus 1/NAD* at varied levels of NADH gave highly non-linear curves. At levels of NAD ÷ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD* showed NADH gave linear uncompetitive inhibition of UDPG if NAD ÷ was saturating. However, at levels of NAD ~ above 0.10 mM, NADH became a competitive inhibitor of NAD* (parabolic curves) and when NAD ÷ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD* was n o t saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD ÷, which is reduced and released. A second mol of NAD ÷ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release o f the second mol of NADH but before the release o f UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting * This work is Paper 9213 of the Scientific Journal Series, Minnesota Agricultural Experiment Station. ** To whom all inquiries and reprint requests should he adressed. Abbreviations: UDPGIcUA, UDPglucuronic acid: UDPGlc-6-CHO, UDP-c~-D-gluco-hexodialdose.

26 step in the overall reaction. The discontinuity in the velocities observed at high NAD ÷ concentrations is apparently caused by the binding of NAD ÷ in the active site after the release o f the second mol of NADH, eliminating the NADH inhibition when NAD ÷ becomes saturating.

Introduction

The formation of UDPGlcUA from UDPG is catalyzed b y UDPglucose dehydrogenase (UDPglucose: NAD ÷ 6-oxidoreductase, EC 1.1.1.22) in an irreversible reaction in which 2 mol of NAD ÷ are converted to N A D H [1,2]. The reaction has been demonstrated to involve the reversible formation of UDPGlc6-CHO from UDPG, followed by the irreversible formation of the corresponding acid [3]. The intermediate aldehyde remains tightly b o u n d to the enzyme surface during the course o f the reaction [ 1]. There is considerable evidence that this enzyme possesses an essential thiol group [4--7] and b o t h the overall irreversibility of the reaction and the tight binding of the aldehyde intermediate can be rationalized b y invoking its function. If the essential thiol group functioned to form a thiohemiacetal with the intermediate aldehyde, then the strong binding of the aldehyde is explained. In addition, it is well established that thiol esters have large negative free energies of hydrolysis [9] and the hydrolysis of the resulting thiol ester would account for the overall irreversibility of the reaction. Such a mechanism has been proposed as being general for aldehyde dehydrogenases [ 10], has been verified in the case of glyceraldehyde-3-phosphate dehydrogenase [11] and has recently been demonstrated in the case of the present enzyme [8]. The purpose of the present work was to determine if the kinetics of the UDPglucose dehydrogenase reaction are compatible with the proposed mechanism. Some inhibition studies have been reported previously. UDPGlcUA has been demonstrated to be competitive with UDPG [2,12,13]. UDPGlcUA is non-competitive with NAD ÷ [2,11] and NADH is uncompetitive with UDPG in the case o f enzyme from Escherichia coli [12]. NADH is reported to be competitive with NAD ÷ [2,13,14]. Materials and Methods

Enzymes and chemicals. UDPG dehydrogenase was purified from bovine liver to a specific activity o f 0.8 unit/rag by the method of Straw [5] with the exception that the extraction solution and the buffers, other than that used in the ethanol fractionation, contained 2 mM EDTA and 10 mM ~-mercaptoethanol. UDPG and UDPGlcUA were obtained from Sigma and NAD ÷ and NADH from P and L Biochemicals. Enzyme assays. All rate measurements were performed at 37°C in a total volume of 0.5 ml. Glycine/NaOH buffer (38 pmol, pH 8.7), 0.5 pmol of neutralized c y s t e i n e . HC1 and 6 - 1 0 -3 unit of UDPG dehydrogenase were incubated for 1 min, at which point a mixture of the substrates and products in distilled water (adjusted to pH 8.0) were added to start the reaction. One unit of enzyme is defined as that amount which will produce 1/~mol of UDP-

27 GIcUA per min under the above assay conditions and in the presence of I mM NAD ÷ and 1 mM UDPG. All reaction velocities are expressed as #M NADH produced per min for the stated reaction mixture. All reaction rates were measured spectrophotometrically by following NADH production at 340 nm using a General Medical and Atomic Energy Commission (GEMSAEC) autoanalyzer (Electro-Nucleonics, Fairfield, N.J.). The GEMSAEC consists of a combination centrifuge, spectrophotometer and computer which carries out fifteen assays simultaneously and provides a numerical printout of the absorbance versus time. For this work readings were taken every 15 s for 5 min. An extinction coefficient of 6.3 • 103 M -1 • cm -~ at 340 nm was assumed for NADH [15]. Treatment o f kinetic data. Through the use of the theory and nomenclature described by Cleland [16--18], initial velocity and product inhibition studies were used to obtain information regarding the sequence of addition of substrates and release of products from UDPG dehydrogenase during catalysis. Rate equations and inhibition patterns were determined for ter-ter and bi-uniuni-bi ordered or random mechanisms both with and without separate rate constants for the hydrolysis of a thiol ester in the mechanism, using equations and techniques described by Plowman [ 19 ]. Initial estimates of kinetic parameters were obtained by least squares plots followed by replots to isolate individual terms in the equation, and then solution of the terms for numerical values of parameters. These estimates were improved by a program involving a Newton-Raphson iteration scheme [20] that fitted all of the parameters simultaneously to the data. Curves in all figures were drawn by linear or quadratic least squares programs. Results

Initial velocity studies. Initial velocity studies were carried out as a function o f NAD ÷ and UDPG concentrations. The enzyme preparation was shown to be free o f inhibitors by demonstrating that enzyme concentration multiplied by time equalled NADH production for 5, 10, 20 and 40 pl of enzyme with 0.25 mM NAD + and 0.10 mM UDPG. Representative initial velocity data are shown in Fig. 1. The intersecting pattern is characteristic of either a random or an ordered sequential mechanism. In Table I the kinetic parameter terms which can be evaluated from initial velocity studies alone are listed. N A D H inhibition. Of particular interest in determining the mechanism are the patterns of NADH inhibition. Because NAD ÷ combines at two distinct points in the reaction sequence, it would not be surprising to find parabolic inhibition by NADH. Because inhibition by NADH has been shown to be competitive only for a narrow range o f conditions [2,13,14], and because the data in some cases appear slightly parabolic [2,13], inhibition by NADH was examined thoroughly. Webb [21] has shown that if a reaction product is an inhibitor and its Ki is small, the reaction rate may drop very rapidly after the reaction has started. Use of the GEMSAEC autoanalyzer allowed both rapid, reproducible mixing and measurement of small variations in absorbance (0.0002). Figs. 2a and 2b show plots with NAD ÷ as the variable substrate and NADH as the changing fixed inhibitor. In Fig. 2a, NAD ÷ concentration was

28

0.3r ,/1OO,

O,

1/UDPG 2 o o j 8 (raM-') sS sS sS •

o.,

100

~>o

,4" O.1~,4/~:fs,,O,."*~ " o A.o

A-o"

1 O.O1

, ,.o

~I/ 0

I00

200

UDPG (mM -1)

s• 0.4

•

~g/" /

5o

#S

• • " 0.2

L

'

210

'

4Jo

1/NAD + (mM-') Fig. 1. Initial v e l o c i t y o f U D P G d e h y d r o g e n a s e a c t i v i t y w i t h N A D + as t h e variable s u b s t r a t e a n d U D P G as t h e c h a n g i n g f i x e d s u b s t r a t e . T h e assay is d e s c r i b e d u n d e r Materials a n d M e t h o d s . T h e r e c i p r o c a l o f initial v e l o c i t y o f N A D H f o r m a t i o n , in #M p e r rain, is p l o t t e d as a f u n c t i o n o f t h e r e c i p r o c a l of t h e c o n c e n t r a t i o n o f N A D +, in m M , in t h e p r i m a r y plot. T h e slopes a n d i n t e r c e p t s are r e p l o t t e d as a f u n c t i o n o f t h e r e c i p r o c a l o f U D P G c o n c e n t r a t i o n in m M in t h e s e c o n d a r y plot.

varied from 0.1 to 2.0 mM. The pattern shows parabolic competitive inhibition. Similar results were achieved at 0.05, 0.025, and 0.0167 mM UDPG. In Fig. 2b, NAD ÷ concentration was varied from 0.01 to 0.05 mM. The pattern indicates parabolic non-competitive inhibition. Similar results were achieved at UDPG concentrations of 0.05, 0.025, and 0.0167 mM. TABLE

I

KINETIC

PARAMETERS

OF UDPG

DEHYDROGENASE

FROM

INITIAL VELOCITY

STUDIES

Values were calculated from replots in Fig. 1 based o n Eqn. 1, where product concentrations are all zero. Calculations were d o n e using the m e t h o d of Elmore et al. [33]. V = 18.9 + 1.9/~M/min (for the assay conditions) KNAD1 KUDGP KiUDG

+ KNAD2 + KNAD3 = 49.9 +- 7.9/~M = 24.6 +- 3.7 # M P , KNAD1 = 1 1 9 1 +- 3 0 2 / ~ M 2

where 1 1 1 l/v=-+--+kS k9 k13 KUDPG V

1 kl

k4 + k 6 KNAD1 = _ _ k3k5 k8 + k9 KNAD2 =

kTk9

k16 + k17 KNAD3 - _ _ klsk17 k2 KiUDP G = __ kl

29

1.C

1/v

05-

~

o o

A

t

i

i

i

i

i_,

5 I / N A D + (m M

mM NADH 0 . 2 0

mMNADH 0.50 O.10 o O.10 0.05

0.05

0 0

,

~

,

i

o

I0

i

i

i

k

i

=

50

tOO

1/NAD + (raM-;)

-I)

Fig. 2. P r o d u c t i n h i b i t i o n o f U D P G d e h y d r o g e n a s e b y N A D H w i t h N A D + as t h e v a r i a b l e s u b s t r a t e a n d U D P G a t f i x e d , n o n - s a t u r a t i n g c o n c e n t r a t i o n s . T h e assay is d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . T h e r e c i p r o c a l o f t h e v e l o c i t y o f N A D H f o r m a t i o n in # M p e r r a i n is p l o t t e d as a f u n c t i o n o f t h e r e c i p r o c a l o f N A D + c o n c e n t r a t i o n in raM. T h e c o n c e n t r a t i o n o f U D P G is 0 . 1 0 raM.

Figs. 3a and 3b are plots of the intercepts in Fig. 2 versus the reciprocal of UDPG concentration. When the intercepts were extrapolated from low NAD ÷ concentrations, NADH gave uncompetitive inhibition of UDPG when NAD ÷ was saturating. In Fig. 3b, where the intercepts were extrapolated from high B

A

0.2

0.1

/ '

~,

' ,,~, ' 6'0 1/UDPG (raM -1)

A

i

80

'

mM NADH 0.20

~ ~uQ.O.8~

0.~0 .....o0.05

O.4

~ '

2'0

o

' 4'0 1/UDPG (raM -~)

'

i

60

F i g . 3. E x t r a p o l a t e d i n t e r c e p t s w h i c h r e p r e s e n t s a t u r a t i o n w i t h N A D + s u c h as t h o s e in Fig. 2, are p l o t t e d as a f u n c t i o n o f t h e r e c i p r o c a l o f U D P G c o n c e n t r a t i o n a t f i x e d levels o f N A D H . P o i n t s in A w e r e e x t r a p o l a t e d f r o m c o n c e n t r a t i o n s o f N A D + b e l o w 0 . 0 5 m M as in Fig. 2b. P o i n t s in B w e r e e x t r a p o l a t e d f r o m c o n c e n t r a t i o n s o f N A D + a b o v e 0.1 m M , c o n t a i n i n g t h e f o l l o w i n g c o n c e n t r a t i o n s o f N A D H : X, 0 . 2 0 m M ; ~, 0 . 1 5 r a M ; e , 0 . 1 0 r a M ; o, 0 . 0 2 5 r a M ; &, 0 raM.

30 N A D ÷ c o n c e n t r a t i o n s , n o i n h i b i t i o n o f U D P G a d d i t i o n was o b s e r v e d w h e n N A D ÷ was saturating. N A D H gave n o n - c o m p e t i t i v e i n h i b i t i o n o f U D P G . F r o m least squares slope a n d i n t e r c e p t r e p l o t s g i s = 14 and 30 pM N A D H w h e n N A D + c o n c e n t r a t i o n was held c o n s t a n t at 0 . 0 1 6 7 and 0 . 1 3 3 mM, respectively, a n d Kii = 80 pM N A D H at b o t h N A D ÷ c o n c e n t r a t i o n s . T h e r a n g e o f U D P G c o n c e n t r a t i o n was 0 . 0 1 6 7 - - 0 . 1 0 m M and o f N A D H 0 - - 0 . 2 0 mM. UDPGlcUA inhibition. U D P G I c U A was c o m p e t i t i v e versus U D P G at 0 . 2 5 and 0 . 0 6 7 m M N A D + w i t h K i = 95 #M U D P G l c U A . U D P G was varied f r o m 0.01 t o 0 . 1 0 m M and U D P G l c U A f r o m 0 t o 0.33 mM. U D P G I c U A was n o n - c o m p e t i t i v e versus N A D ÷. At 0.1 m M U D P G , Kii = 0.55 m M and K~ = 0 . 1 3 m M U D P G I c U A . At 0.01 m M U D P G , Kii = 0 . 1 8 m M and Kis = 0 . 0 6 m M U D P G l c U A . T h e range o f N A D + c o n c e n t r a t i o n was 0 . 0 3 3 - 0 . 2 0 m M and o f U D P G I c U A was 0 - 0 . 3 3 mM. This is c o n s i s t e n t w i t h an o r d e r e d m e c h a n i s m in w h i c h U D P G adds first, b u t n o t w i t h o n e in w h i c h N A D ÷ w o u l d add first and U D P G l c U A w o u l d act as a d e a d e n d i n h i b i t o r w i t h t h e e n z y m e • N A D ÷ c o m p l e x . T h e l a t t e r w o u l d result in an u n c o m p e t i t i v e i n h i b i t i o n p a t t e r n .

Discussion A m e c h a n i s m b y w h i c h all t h e p a t t e r n s c a n b e e x p l a i n e d is s h o w n b e l o w , w h e r e A = U D P G , B = N A D +, P = N A D H , R = U D P G l c U A , A' = U D P g l u c o s e 6a l d e h y d e , and E R ' is t h e t h i o l ester b e t w e e n t h e e n z y m e and U D P G l c U A .

A

B

P

B

kl~k 2

k3~k4

k$f k6

k7~ k8>

E

EA

(EAB-EA'P)

EA'

B

P

k~s~k~6/ER'B __ ktTIhis

kgtk~o

/ ER'

(EA'B-ER'P)

B

~xl

R k~3t~14

hl~

ER

)

E

T h e r a t e e q u a t i o n f o r this m e c h a n i s m is given in E q n . 1.

2/v

=1

+1

+ 1

ks

1 1

ks

+ k9

1 + k2ks + k2k4 _ _1

k,k3k,

k2k4k14 + k2ksk14 R k6ks + k6k9 - -P+ k,k3ksk,3 AB + ksk~k9 B

AB

+ _ k14 _

R

klk13 A

k2k4k6(ks + k9)

P

k,k3ksk7k9

AB 2

k2k4k6k14(]~rl~ + k9) P R + X I 1 + kloP + ksklo - + klk3kskTk9k13 AB 2 " ~ kTk9

P + keksklo p2 + k4keksklo p2 B kskTk9 B k3kskTk9 B 2

k2k4k6ksk10 - -

k2k4k6ksk,okl4 P2R~ + y~k4 + ks I + k4k6(ks + k 9) p']

k,k3hskTk9 A B 2

kl~3k$~7k~gk13 -~...J

k-k-~-~ B

k3ksk,k9

B:J

31 where X =

k16 + k17 k l l ( k l 6 4- k17) 4- k,sklTB

kllk17 + klsklTB Y = kll(k16 + kiT) 4- klsklTB

(1)

All o f the kinetic patterns observed are compatible with the above mechanism and equation. A characteristic of the ordered addition of substrates is that NADH becomes an uncompetitive inhibitor versus UDPG at saturating levels o f NAD ÷ because the addition o f NAD ÷ becomes irreversible. The relevant equation which can be derived from Eqn. 1, is:

2/v=

1 +1+

k9

1

1

1 1

kl0P

The agreement of Fig. 3 A with Eqn. 2 confirms the ordered addition with U D P G adding first. However, at high levels of N A D + the extrapolated intercepts show no inhibition by N A D H , as show in Fig. 3B. For all inhibition to be overcome, both steps at which N A D H is released must be irreversible.The proposed mechanism accounts for this by the additional binding of N A D ÷ after the release of the second N A D H . This binding of N A D + is competitive with N A D H , but otherwise does not participate in the reaction. The relevant equation to Fig. 3B is shown below. 1

1

1

A 1

2 / v = ~ + ~ + ~ + k~A

(3)

The overall reaction is irreversible [1,2,3]. The kinetic data firmly support the occurrence o f an irreversible step after the second release o f NADH, b u t before the release o f UDPGlcUA. All o f the other steps must be reversible for the mechanism to fit the data. No other mechanism could be derived which is compatible with the patterns observed. It is n o t e w o r t h y that this confirms the observation that both redox steps are reversible [7]. It is postulated that this irreversible step (k~2 - k 18 ~ O) is associated with the hydrolysis o f a thiol ester as has been suggested for this and for other aldehyde dehydrogenases [8,10,11, 22,23]. The thiol ester hydrolysis is not only the irreversible step, b u t also the ratelimiting step o f the reaction. From the data in Fig. 3 and Eqns. 2 and 3, it can be calculated that kl~ - 17/zM NADH/min and that V = 17/zM NADH/min. Therefore k , , the thiol ester hydrolysis, must be the rate-limiting step in the reaction sequence, equal to the observed maximum velocity. This idea is supported b y several observations. The rate-limiting step occurs after the formation o f UDPGIc-6-CHO [ 3 ]. The second redox reaction is readily reversible [ 7 ], indicating that it involves a thiol ester, k n is similar in magnitude to the rate constants for the hydrolysis o f thiol esters between papain and hippuric acid [3O]. T w o other 4-electron dehydrogenases, hisitidinol dehydrogenase and hydroxylmethylglutaryl-CoA reductase, have been shown to require thiol groups

32 [31,32]. In the case of histidinol dehydrogenase the thiol group is a part of the enzyme's structure and could well function in a fashion similar to that postulated here. For hydroxymethylglutaryl-CoA reductase the thiol is provided by CoASH, which functions exactly as in the manner postulated here for the essential thiol o f UDPG dehydrogenase. The ordered addition of the first two substrates demonstrated here for UDPglucose dehydrogenase is well established for other dehydrogenases. In the case o f horse liver alcohol dehydrogenase [24,25], pig heart malate dehydrogenase [26], beef heart and muscle lactate dehydrogenase [27], beef liver L-glutamate dehydrogenase [28], and horse liver aldehyde dehydrogenase [22], N A D ÷ binds first. For yeast aldehyde dehydrogenase, the substrate rather than NAD ÷ binds first [27], as is the case with the present enzyme.

Acknowledgements We thank Dr. Philip Blume of the University of Minnesota for assistance in the use o f the GEMSAEC autoanalyzer in his laboratory. This work was supported by grant GM 0 9 2 9 3 from the National Institutes of Health. References 1 Stromingex, J.L., Kalckar, H.M., Axelrod, J. and Maxwell, E.S. (1954) J. Am. Chem. Soc. 76, 6411-6412 2 Molz, R.J. and Danishefsky, I. (1971) Biochim. Biophys. Acta 250, 6--13 3 Nelsestuen, G.L. and Kirkwood, S. (1971) J. Biol. Chem. 246, 3828--3834 4 Maxwell, E.S., Kalckar, H.M. and Strominger, J.L. (1956) Arch. Biochem. Biophys. 65, 2--10 5 Straw, T.E. (1969) Ph.D. Thesis, University of Minnesota 6 Gainey, P.A., Pestell, T.C. and Phelps, C.F. (1972) Biochem. J. 1 2 9 , 8 2 1 - - 8 3 0 7 Ridley, W.P. and Kirkwood, S. (1973) Biochem. Biophys. Res. Commun. 54, 9 5 5 - - 9 6 0 8 Ridley, W.P., Houchins, J.P. and Kirkwood, S. (1975) J. Biol. Chem. 250, 8761--8767 9 Lowe, G. and Williams, A. (1965) Biochem. J. 9 6 , 1 9 4 - - 1 9 8 10 J a k o b y , W.B. (1963) in The Enzymes (Boyer, P.O. and Lardy, H.A., eds.), Vol. 7, pp. 203--221, Academic Press, New York 11 Harris, J.I., Merriwether, B.P. and Park, J.H. (1963) Nature 198, 154--157 12 Neufeld, E.F. and Hall, C.W. (1965) Biochem. Biophys. Res. Commun. 19, 456--461 13 Schiller, J.G., Bowser, A.M. and Feingold, D.S. (1973) Biochim. Biophys. Acta 293, 1--10 14 Zalitis, J. and Feingold, D.S. (1968) Biochem. Biophys. Res. Commun. 31,693---698 15 Racker, E. (1955) Physiol. Rev. 35, 1--56 16 Cleiand, W.W. (1963) Biochim. Biophys. Aeta 67, 104--137 17 Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 173--187 18 Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 188--196 19 Plowman, K.M. (1972) Enzyme Kinetics, McGraw-Hill, New York 20 Caxnahan, B., Luther, H.A. and Wiles, J.O. (1969) Applied Numerical Methods, p. 319, J o h n Wiley, New York 21 Webb, J.L. (1963) E n z y m e and Metabolic Inhibitors, Vol. 1, p. 144, Academic Press, New Y ork 22 Feldman, R.I. and Weiner, H. (1972) J. Biol. Chem. 2 4 7 , 2 6 7 - - 2 7 2 23 Segal, H.L. and Boyer, P.O. (1953) J. Biol. Chem. 204, 265--281 24 Theoren, H. and Chance, B. (1951) Acta Chem. Scand. 5, 1127--1144 25 Wratten, C.C. and Cleland, W.W. (1963) Biochemistry 2, 935--941 26 Raval, D.N. and Wolfe, R.G. (1962) Biochemistry 1, 263--269 27 Takenaka, Y. and Schwert, G.W. (1966) J. Biol. Chem. 223, 157--170 28 Frieden, C. ( 1 9 5 9 ) J. Biol. Chem. 234, 2891--2896 29 Bradbury, S. and J a k o b y , W.B. (1971) J. Biol. Chem. 246, 1834--1840 30 Lowe, G. and Williams, A. (1965) Biochem. J. 96, 199--204 31 Loper, J.C. (1969) J. Biol. Chem. 243, 3 2 6 4 - - 3 2 7 2 32 Fimognari, G.M. and Rodwell, V.W. (1965) Biochemistry 4, 2 0 8 6 - - 2 0 9 0 33 Elmore, D.T., Kingston, A.E. and Shields, D.B. (1963) J. Chem. Soc. 2070--2078