Biochimie ( ! 991 ) 73, 611-613 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris
611 Short communication
Preliminary studies on quinoprotein glucose dehydrogenase under extreme conditions of temperature and pressure J F r a n k J z n ' , J A Duine~, C Balny2* tDepartment of Microbiology and Enz3'mology, Delft Universit~ ~/'Techmdogy, ,lulianalaan 67. NL-2628 BC. Delj?, The Netherlatids: "-INSERM Unit¢~ 128. Site du CNRS, BP 5051. 34033 Monqwllier Cedex 1. France (Received 23 October 1990: accepted 17 January 1991)
S u m m a r y - - The kinetics of the reduction of the quinoprotein glucose dehydrogenase by substrate were studied as a function of 3 parameters: pressure (1-1000 bar), temperature (down to -25°C) and solvent (water and 40% dimethyl suifoxide, DMSO) using a high-pressure low-temperature stopped-flow apparatus. A 2-step formation of the reduced enzyme by its substrate (xylose), was observed. A rapid equilibrium described by the constant K~ was followed by a slower process described by the constants k, and k ,. By using the transition state theory, the thermodynamic quantities AV - (activation volumes) were determined for these various kinetic constants under different experimental conditions. The results are discussed in terms of conformationai change and solvation effect on the protein shell, and compared with results obtained for other systems as the 2-step formation of horseradish peroxidase compound I. quinoprotein
glucose dehydrogenase / pressure
effect / t e m p e r a t u r e effect / solvent effect / stopped-flow
During the past 10 years it has become clear that several periplasm-located bacterial dehydrogenases are quinoproteins, ie enzymes containing 2,4,7-tricarboxy- IH-pyrrolol2,3-Jlquinoline-4,5-dione (PQQ) as a cofactor [1 ]. Mechanistic studies on these quinoproteins have been concentrated mainly on methanol dehydrogenase [21. However, the poorly understood role of ammonia as an activator and the failure to prepare a reconstitutable apo-enzyme are serious drawbacks associated with this enzyme. Quinoprotein glucose dehydrogenase seems more attractive in these respects: no activator is required and a reconstitutable apo-enzyme is available to study factors relevant for binding and activity of the cofactor by way of studies with analogues. Two quite different types of glucose dehydrogenase are known : the soluble type (EC 22.214.171.124) present in Acinetobacter calcoaceticus LMD 79.41 and a membranebound form, distributed in many Gram-negative bacteria. The soluble glucose dehydrogenase (GDH) used in the present study is the most attractive, since it does not require the presence of complicating deter*Correspondence and reprints Abbreviations: GDH, quinoprotein glucose dehydrogenase; DMSO, dimethyl sulfoxide
gents and large amounts of enzyme are available via recombinant DNA teclmiques [4, 51. At room temperature the reduction of oxidized GDH by glucose or even xylose is too fast to accurately determine the thermodynamics of the reaction pathway. As previously shown for other enzyme systems, we have used the cryo-baro-enzyme approach to obtain more insight into the mechanism of action of the enzyme. To our knowledge GDH is the first quinoprotein to be experimented with under pressure and at sub-zero temperatures. The basic data obtained under these extreme conditions may contribute to a better understanding of catalysis by quinoproteins and assist future biotechnological exploitation of these enzymes. Materials and Methods Materhds All chemicals were obtained from E Merck. The anti-freeze solvent chosen was dimethyl sulfoxide (DMSO) in a concentration of 40% (v/v). The polyols generally used lor cryoenzymology (eg ethylene glycol) were unsuitable because they behave as substrates for the enzyme. Tris-HCl was chosen as a buffer since its hydrogen concentration is almost pressureindependent [61. Glucose dehydrogenase, a gift from BW Groen, was prepared as described [51.
JF Jzn et al
The reduction of oxidized GDH (2.1 mM) with D-xylose in concentrations ranging from 0.1-100 mM was followed at 337 nm. The effect of pressure was studied with 1.125 mM xyiose. Experiments at atmospheric pressure and at room temperature were made using a Hi-Tech Scientific stoppedflow spectrophotometer, model PQ/SF 53. Kinetic measurements at high pressures (up to 1 kbar) and low temperatures (down to -25°C) were peformed using a high-pressure stoppedflow apparatus developed in the laboratory of one of our team (CB) [7. 81. Reaction curves were fitted using an Apple II micro-computer and the KINFIT program. Data concerning the pressure dependency of various kinetic constant were considered within the transition state theory  and activation volumes AV and AV -+were calculated according to previously published procedures [ 10l.
Results a n d Discussion
Two-step reaction process studied using soh,ent and temperature effects The simplest fundamental form of an enzyme reaction is a 2-step mechanism. The first steps of the reaction of oxidized G D H with substrate can be postulated as follows: K~ GDH ox + Xyl ~ [GDH,Xyl]
k_, " GDHred.Xylonolacton k_2
(scheme 1) In order to determine the constants, we have studied /~"
. . . .
! . . . . . . . . . .
. . . . .
different experimental conditions. One can obtain KI and kz, or only the second-order constant k÷ if a saturation plateau cannot be attained (reaction too fast, or K~ too small). If ko~ follows a hyperbolic relationship and if [S] > [E]:
kob~ = kz.K,.[S]/(K,.[S] + 1) + k_z
Table I. Values for k÷, K~ and k2 under different experimental conditions (SD + 10%). nd: not determined.
k+ = Ki.k2 = k,,hJlS]
40% DMSO -14
Even though measured at a different temperature, the presence of 4 0 % D M S O does not appreciably affect the value for K~ (see table I). D M S O being a potent 'water structure breaker' this small effect is of interest since it implies that the binding of substrate is insensitive to the dielectric cor;stant of the solvent environment and apparently does not involve electrostatic interactions. A similar conclusion was reached for the formation o f compound I in the horseradish peroxidase-peroxide reaction [ 13].
High pressure kinetic experiments Because the dead time o~,: our high-pressure stoppedflow equipment is --- 5 ms , it was impossible to record activation volumes in buffered water for K~ and k2 respectively (the reactions were too fast). Under this experimental condition, only the activation volume for the composite constant k+ = K~.k2 was obtained (slope of the dependence o f kob., vs xylose concentration) W~ c n m n n r o H tho AIL/'~tv ~ l n o e ~ h t ~ i n , ~ d
under different experimental conditions (see table II). Plots of In k+ vs pressure, were linear, indicating that no change of rate-limiting step at high pressure occurs , and the slopes of the lines give AV ~, assuming R = 82 cm3.atm.K-~.mol-I. However, we have pointed out previously that the variations observed in L~V +~in different media (the temperature being the same) suggest that solvent reorganization is an important
k+ = k2-K, = kobJ[S] At room temperature in water buffered solution as well as at - 1 4 ° C in 40% D M S O , we have obtained evidence that enzyme reduction involves 2 steps: a rapid equilibrium (K~) followed by a slower process described by k2 and k_2 (see table I). In addition, extrapolation of kobs to zero concentration of xylose gave a value close to zero which implies a very small k : in agreement with the high redox potential of G D H (+ 50 m V ) compared to that of the assumed redox potential of xylose (unpublished observations) which must be not very different from that for glucose/glucanolacton: --400mV [ 12].
Table I. Activation volumes (in ml.mol-l) for k÷, K~ and kz under various experimental conditions (means + SD). nd: not determined.
Temp l °C1
Nature of kinetic constants KI k2 k+ IAVI laV~l IAV~1
-7.8 + 0.5
-1.3 + 1
-7.4 + 4
20.8 + 41
3.4 + 1
Temperature and pressure on quinoprotein dehydrogenase factor w h i c h , associated with a c o n f o r m a t i o n a l c h a n g e (see below) could drive the response o f the s y s t e m . The apparent AV * m e a s u r e d is the sum o f several c o m p o n e n t s : binding, c o n f o r m a t i o n a l c h a n g e and solvation [ 10]. W e were able to d e t e r m i n e the effect o f pressure on K~ and k, o n l y at sub-zero temperatures. T a b l e II s h o w s data o b t a i n e d at - 1 4 ° C in 4 0 % D M S O . It appears that the absolute v a l u e o f the activation v o l u m e o b s e r v e d for step 2 is rather large c o m p a r e d to that for step 1 : 2 0 . 8 + 4 and - 7 . 4 + 4 ml.mol-~ respectively. T h i s observation m u s t be c o m p a r e d with the data a l r e a d y p u b l i s h e d for the f o r m a t i o n o f horseradish p e r o x i d a s e c o m p o u n d I [ 13]. In the latter case there was no e v i d e n c e that the substrate induced a c o n f o r m a t i o n a l change, since the spectral c h a n g e s observed upon binding; o f p e r o x i d e could h a v e originated from s o m e m o v e m e n t s w i t h i n the peroxide molecule. T h i s m a y also o n l y i n v o l v e the h e m e m o i e t y o f the protein . Despite the lack o f inform a t i o n on the d y n a m i c s o f the protein it could be postulated that the large AV z-+recorded is associated with a s u b s t r a t e - i n d u c e d c o n f o r m a t i o n a l c h a n g e in the e n z y m e . In the a b s e n c e o f spectral modification, it is not possible to d e t e r m i n e w h e t h e r the reaction follows the predicted ' i n d u c e d - f i t ' theory. Further e x p e r i m e n t s , m a i n l y as a function o f temperature, are n e e d e d to substantiate the present results. Nevertheless, this is another e x a m p l e in w h i c h AV * is a t h e r m o d y n a m i c value w h i c h varies d r a m a t i c a l l y and e v e n c h a n g e s sign as a function o f the e x p e r i m e n t a l conditions (eg the effect o f temperature on AV L; see table I). A l t h o u g h this still leaves o p e n the question o f the precise p h y s i c a l m e a n i n g o f AV *, data obtained under pressure, however, reveal a d y n a m i c structural flexibility o f the e n z y m e , largely controlled by the nature o f the m e d i u m .
Acknowledgments This work was supported by grants from La Fondation pour la Recherche M6dicale Franqaise and the Direction des Recherches, Etudes et Techniques (DRET No 89/037). We thank the French Minist~re des Affaires Etrang~res for its encouraging financial support.
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