Evidence of NADH channeling between dehydrogenases

Z theo~ BioL (1991) 152, 103-107

Evidence of NADH Channeling between Dehydrogenases

Ovfidi (1991) has written a thorough, thoughtful, and provocative review of metabolite channeling in my opinion. She describes the evidence for NADH channeling as "Controversial Data" since some criticisms of channeling have been recently published, (e.g. Chock & Gutfreund, 1988; Kvassman & Pettersson, 1989). Yet as specified below, there are extensive data indicating N A D H channeling, and the results for only one dehydrogenase pair have been disputed with experiments. The most pertinent and convincing data indicating N A D H channeling are obtained by the simple, but rigorous "enzyme buffering" method. This method is one of the few applicable to enzymes, which are not extensively associated in vitro (see below). If the dehydrogenases remained extensively associated in the presence and absence of reaction intermediates, the competing reaction test would be the method of choice to test for substrate channeling (Datta et aI., 1985). With the enzyme buffering method, E2 is maintained at its conventional assay concentration (sub-~g m1-1 or riM). First the Km and Vm of the E2 reaction for N A D H are determined in the absence of E1 (at a fixed cosubstrate concentration). Next, similar measurements on this same reaction are repeated, but in the presence of very high concentrations of Et (10-200 p~M). This E~, in excess of the total N A D H present, binds most of the NADH. The cosubstrate of Et is not present, so Et is only "buffering" N A D H - - i t is not catalyzing any reaction by itself. Independent measurements establish the dissociation constant Kd for E~-NADH. Since [E2] is only 10-4-10 -5 of l e t ] , the equilibrium between Et and N A D H is not altered in the subsequent kinetic measurements. Thus, the equilibrium concentration of free NADH is known at each experimental point of the velocity vs. [ E r N A D H ] data. Therefore, if unbound (free) N A D H is the only enzymatically active form of NADH, the reaction velocity expected is that calculated as l)¢alc= Vm[NADH]I/ (Kin + [NADH]s), where the subscript f represents free NADH. In virtually all cases where E~ and E2 have opposite chiral specificity for the C4-H of NADH, experimental velocities Vexp can be obtained which are ten to 20 times vca~c. This was first demonstrated in the 1950s for several different NADH acceptor enzymes using glyceratdehyde-3-phosphate dehydrogenase (GAPD) as the donor enzyme (Coil et al., 1950; Mahler & Elowe, 1954; Nygaard & Rutter, 1956). These observations were forgotten until Srivastava & Bernhard (1985a) greatly expanded the data using many different donor and acceptor enzymes, clarified experimental considerations and interpretations, and offered a plausible molecular interpretation of the chiral specificity of the process (Srivastava & Bernhard, 1985 b). We and others have subsequently used this method (Fahien et aL, 1989; Ushiroyama et al., 1991). Considering the carefulness of these experiments, the number of independent studies and examples (~20), the simple yet rigorous nature of the 103

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method, and the remarkable insensitivity to experimental errors of the conclusions from properly designed experiments (Fukushima et al., 1989; Ushiroyama et al., 1991), these results simply cannot be ignored. Significantly, Vexp= Vca~cwithin a 10% standard deviation whenever E~ and E2 are of the same chiral specificity (about eight examples). I refer to these experiments below as "control experiments". These controls demonstrate the lack of artifacts with the enzyme buffering method. Assuming the validity of the enzyme buffering results, I know of only two possible explanations for vexp>>vc,~c. One is that an E~-E2 complex is formed with an enormously lower Km (90-fold in one of our studies) than that of E2 ("reactive complex" mechanism). The other explanation would be to assume that the only other form of NADH present, namely E~-NADH, is enzymatically active as well as free NADH ("channeling" mechanism). The available evidence strongly favors the second interpretation (Ushiroyama et al., 1991). The model equation for the proposed channeling scheme is simply the initial velocity equation of an enzyme E2 that can use two forms of its substrate, free and enzyme-bound NADH in this case. It is then a simple matter to calculate the fraction of total flux passing through either the classical or channeled path for given concentrations of total E~ and NADH (Ushiroyama et al., 1991). This model equation is phenomenological. Beyond the verifiable Michaelis dependence on [NADH] and [E~-NADH], no assumptions of kinetic mechanisms are required for validity of this equation, not even knowledge of whether El dissociates from E 2 for each reaction turnover or not (Batke, 1989). This equation predicts that the vast majority of the reaction flux passes through the channeled path with concentrations of NADH binding sits and total NADH expected in vivo (Ushiroyama et al., 1991). The dominant factor in determining the flux ratio through these paths is the ratio of enzyme-bound to free NADH, not the kinetic constants (Fukushima et al., 1989; Ushiroyama et al., 1991). The assumption that the kinetic constants of the channeled path have to be much more favorable than those of the classical path is unnecessary and cannot be justified. No equilibrium association of E r N A D H with E2-NADH (in the absence of the E 2 cosubstrate) was detected for the three enzyme pairs with which this was sought (Fukushima et al., 1989; Nygaard & Rutter, 1956; Wu et al., 1991). It is not difficult to understand how substrate channeling could occur with only transient formation of E~-NADH.E2 (see e.g. Dillon & Clark, 1990; Fukushima et al., 1989). However, saturation kinetics with respect to enzyme bound NADH have been demonstrated in most of these cases. Thus, no matter which interpretation of the above results is assumed (reactive complex or substrate channeling), we must invoke saturation of E2 forms with E~ during the reaction. As described elsewhere (Ushiroyama et aL, 1991), there is good reason to believe that the failure to observe such complexes in the three cases studied is due to the use of NADH concentrations that saturate both enzymes, and/or the lack of both substrates maintaining a favorable steady state of reaction forms. The only experimental studies disputing the above NADH observations (Vexp>> veaj¢) are those of Chock et al. (Chock & Gutfreund, 1988; Wu et aL, 1990). This is primarily with the dehydrogenase pair for lactate and c~-glycerolphosphate (LDH

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and a GPD, respectively).f However, they have not performed any enzyme buffering measurements that are definitive for or against NADH channeling. The vast majority of their experiments measure the rates of N A D H transfer from E1 on E2 or obtain auxiliary data for their interpretation. I shall refer to this as the "transfer rate" method o f testing effects of E~ on the kinetics o f E2. In addition to many other shortcomings, this approach, by their own admission, failed to exclude substrate channeling unless it could be shown that association of E~ a n d / / 2 did not occur. The necessity of such enzyme complex formation during the reaction is apparent from the much simpler and definitive enzyme buffering data. Therefore, I do not think the transfer rate measurements are an adequate test of substrate channeling. For example, in fig. 4 o f their study (Wu et al., 1991), the k~ used to fit the data is not experimentally determined. It was varied to fit the data. Chock & Gutfreund did provide three experimental data points in an inadequately designed enzyme buffering experiment (Chock & Gutfreund, 1988: fig. 5). Two o f these points (added E~) did not decrease free [NADH] significantly and the third point only decreased free [NADH] to ~ K , , . This overlooks the essential requirement for an enzyme buffering test to be able to detect and evaluate channeling, if it exists. That is, enough El must be added to decrease free [NADH] to << Kr,. This reduces v¢a~cso that Vexp>>Vca~¢when significant channeling exists. Instead, they were content to argue that inhibition by the added E~ in their experiment was contrary to NADH channeling (Chock & Gutfreund, 1988). In fact, this inhibition (relative to the Vm for free NADH) is exactly what the NADH channeling model equation (Ushiroyama et al., 1991) and constants predict for this and several other enzyme pairs where both the K,, and V,, o f the channeled path are lower than for the free NADH (Srivastava & Bernhard, 1985a). The reason for their lower K,, of a-glycerolphosphate dehydrogenase for NADH in these enzyme buffering data (Chock & Gutfreund, 1988) has now been identified. This resulted from their use of a much lower cosubstrate concentration (Wu et al., 1991). Corrected for this effect, their Michaelis constant now agrees very well with ours. However, the resulting operative K,, for E2 in their enzyme buffering measurements was 2.5-fold lower than the Kd of E ~ - N A D H . With this condition it becomes essentially impossible to decrease free [NADH] adequately with practical concentrations of E~ to discriminate channeling from a classical reaction. For example, from our more complete data and kinetic constants, it can be shown that 97% of the reaction flux was passing through the channeled path for this point ([NADH]rr,, ~ K,,) of their fig. 5 (Chock & Gutfreund, 1988) (calculation details available upon request). By contrast, the K,, in our lab and that of Srivastava's was about three-fold higher than the Kd (Srivastava et al., 1989). We also used a range of [E~] that ultimately decreased free [NADH] to 14-fold below Ks. In the original study f Theydo refer to ~investigatingthe aldolase/aGPD pair with enzymebufferingmeasurements(Chock & Gutfreund, 1988).No data are presented. However,they again use the erroneouscriterionof inhibition as the evidenceagainst channeling. Also they are content to use a ten-fold range (3-30 p.M) of literature values of K d to claim agreementwith the classical mechanism. No conclusionsfor or against substrate channeling can be obtained from such limited data.

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(Srivastava & Bernhard, 1985a), conditions were even more favorable. The Kd of the d o n o r L D H was 13-fold lower than the K,, of E2 and the free [ N A D H ] was decreased to 43-fold lower than the K,, of E2. These results unequivocally demonstrate the inadequacy of the classical mechanism. Wu et al. (1991) contend that interpretation of enzyme buffering data are subject to large errors, and that only " . . . small inconsistencies between data and theory • . . " result from this method• This is remarkably contrary to the published f a c t s - - s e e also our calculations on the insensitivity of the results to errors ( F u k u s h i m a et al., 1989; U s h i r o y a m a et al., 1991)• Ratios of vexp/Vca,¢ for the system in dispute as high as 11 are given in table II of Srivastava & Bernhard (1985a) alone; i.e. results 110 times larger than errors in the control experiments. Results from two other independent experiments with the enzyme pairs in dispute are given in table 1 of Srivastava et al. (1989)• Results from my lab were not included in the latter publication for lack of space, but were sent to Dr C h o c k in July of 1990, following their presentation at the ASBMB Meeting (Wu et al., 1990). Altogether, these data represent four independent experiments with about 24 data points, all of which exhibit large ratios of V~xp/Vca~¢. These large ratios are in sharp contrast with the control where a G P D is paired with G A P D having the same chiral specificity (Srivastava & Bernhard, 1985a). N u m e r o u s other enzyme pairs with opposite chirality were shown to exhibit similarly large deviations of re×p~ Vca,¢in the study of Srivastava & Bernhard (1985a) alone. For example, the L D H - G A P D pair shows /')exp//')calc values as high as 24 for the forward reaction and 5.2 for the reverse reaction (see also Srivastava & Bernhard, 1959: table IV). These are 240 and 52 times, respectively, the error in control experiments. Their suggestion that the errors in enzyme binding site concentrations could reconcile the data on the L D H - c e - G P D system with the classical path are also unreasonable. First, as I described in my letter to Chock of July 1990, we measure the enzyme binding site concentrations during the fluoresence titrations with N A D H and perform the enzyme buffering experiments with these experimental binding site concentrations• Secondly, such errors are not compatible with the excellent agreement between theory and experiment in the control experiments with these enzymes• Thirdly, the errors p r o p o s e d are not large enough to account for the large yelp~/.)calc observed. Incidentally, errors in the enzyme buffering data cannot be blamed on low kcat values either, since uca,¢ is calculated on the basis of the experimentally determined kcat (Vm) , not a hypothetical m a x i m u m value. We used these enzymes without further purification in order to reproduce the same commercial source and form of enzymes and experimental conditions that they had used in their earlier study (Chock & Gutfreund, 1988)• Their criticism that errors accumulate due to " . . . the m a n y constants i n v o l v e d . . . " is equally invalid• In addition to the E ~ - N A D H dissociation constant, only the K,, and V,, constants in the two kinetic measurements are needed. These are especially easy to obtain since the reactions are extremely favorable for N A D H oxidation• Excellent agreement of all constants with literature values, including those from Chock's lab, are obtained. Again, the consistency of these constants is proven from the control experiments• The n u m b e r of constants needed for their transfer rate method are more numerous, less rigorous, and more difficult to obtain•

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In summary, extensive enzyme buffering data document the inadequacy of the classical mechanism of coupled dehydrogenase reactions with physiological ratios of bound-enzyme and free-NADH (ratios > 20 for most enzymes). Greater than 90% of the reaction flux would be expected to be channeled with these conditions. This is true even when Vm of the channeled path is three-fold lower than that of the classical path (Ushiroyama et al., 1991). Conclusions from properly designed enzyme buffering experiments are remarkably immune to experimental error. Chock et al. (Chock & Gutfreund, 1988; Wu et al., 1991) ignore most of these data and grossly misrepresent the little they do refer to. In addition, they use inadequate methods, including conditions too limited, to test for the effect that they try to exclude. In spite of these negative and inadequate data (one enzyme pair ancillary to glycolysis), they are confident in extrapolating conclusions to all of glycolysis (Wu et al., 1991). Finally, I recommend anyone who wishes to explore these phenomena to consider enzyme buffering measurements. With many, but not all enzyme pairs, the measurements are very simply and rapidly made. The principal precaution for dehydrogenases is the removal of contaminating NADH oxidases that occur in some preparations (Fukushima et al., 1989). I would be happy to share experiences with those who might encounter questions or problems. Grateful appreciation for support (grant OK-88-G-12) from the American Heart Association, OK-Affiliate grant is acknowledged. This is journal article J5937 of the Oklahoma Agricultural Experiment Station. Department o f Biochemistry, O k l a h o m a S t a t e University, Stillwater, O K 74078-0454, U.S.A.

H . O L I N SPIVEY

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