Dec 17, 1991 - activity of rat liver in [1] (Table 2) at 0.5 mM-pyruvate was 970 munits/g. ... The decrement in 14CO2 production from [1-_4C]pyruvate with.
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partly compromised by the formation of the adducts of sample with the sinapinic acid matrix mentioned by Allen et al. The addition of a matrix is essential for analysis by the LDTOF technique to be successful. That there is a further limitation to resolution can be inferred from the Figure where the adduct peaks themselves are only partly resolved from the protonated molecular ion peaks at a mass difference of 206 Da (about 1 in 80 in mass). Allen et al. make no comment on the resolution obtained by ES m.s. on this mixture and all the ES spectra in the paper are presented in a way which does not give the reader the opportunity to assess the resolution. In our ES m.s. data, the a and /8 globins are fully resolved down to the base line and, moreover, there is sufficient resolution available to partially separate, but nevertheless accurately measure, two globins differing in mass by as little as 14 Da at 16 000 (1 in 1 100), for example the two y-globins in foetal haemoglobin with molecular masses of 15995.2 and 16009.3 [2]. The analysis of more than a thousand protein samples in our laboratory has demonstrated the utility of the higher resolution available with ES m.s. because a significant proportion of samples contain closely spaced components which would not be resolved by the LDTOF instrument. In such samples the molecular mass measured by LDTOF would, at best, be an average of the components present. The LDTOF spectra shown in Allen's paper and those also given in a subsequent paper in the same issue [3] indicate performance where the resolution is at least a factor of 10 lower than with ES m.s. These comments are not meant to imply that the LDTOF technique does not have a niche in protein mixture analysis. On the contrary, its reported higher sensitivity, mass range and resistance to the presence of buffers etc. in the sample make it complementary to ES m.s. However, I maintain that [1] presented the comparison between the two techniques in an unbalanced and misleading fashion and hence justifies this response.
single-substrate enzymes such as subtilisin or chymotrypsin this presents no problems, but with two- or three-substrate enzymes such as tyrosyl-tRNA synthetase, glutathione reductase or lactate dehydrogenase it needs to be used with care. In the Dalziel representation [3] of the initial-rate equation (eqn. 1)
Brian N. GREEN
kcat. Km(pyruvate) X Km(coenzyme) To quote, " in evaluating the relative efficiency of an enzyme with alternative coenzymes, the substrate Km must be taken into account, i.e. the ability of the enzyme-coenzyme to bind the
VG Biotech, Tudor Road, Altrincham, Cheshire WA14 5RZ, U.K. 1. Allen, M. H., Grindstaff, D. J., Vestal, M. L. & Nelson, R. W. (1991) Biochem. Soc. Trans. 19, 954-957 2. Green, B. N. et al. (1990) in Biological Mass Spectrometry (Burlingame, A. L. & McCloskey, J. A., eds.), pp. 129-146, Elsevier Science Publishers, Amsterdam 3. Mock, K. K., Davey, M., Stevenson, M. P. & Cottrell, J. S. (1991) Biochem. Soc. Trans. 19, 948-953
Received 17 December 1991
Specificity constants in the context of protein engineering of two-substrate enzymes It has become popular in recent years to combine the two constants of the Michaelis-Menten equation in a ratio, the socalled specificity constant, kcat/Km [1,2]. This is frequently used for comparing the activities of a single enzyme towards two alternative substrates. It has a clear physical meaning, representing the proportionality of rate to substrate concentration at low concentrations (i.e. well below Kn). Thus, with the same amount ofenzyme and equal low concentrations of two substrates (separately), the rates should be in the ratio of their specificity constants. This constant has also been adopted as a convenient way of comparing the activities of protein-engineered mutants with the activity of the corresponding wild-type enzyme. With effectively
e
+
OA
B +AB
v[AAl[B] [A][B]
(1)
Km for substrate A is OA/S00, kca, is 1/bo and the specificity constant for substrate A is simply I/56A. Similarly for B the constant is 1 /qB. It is immediately apparent that both the Km for A and the Vma. must be measured with, or rather extrapolated to, saturating concentrations of B. If the concentration of A is varied only at a single, supposedly saturating, concentration of B it cannot be assumed that the same concentration of B is necessarily saturating for a mutant form of the enzyme. It is nonetheless increasingly common to present only the constants without the evidence for a valid procedure of extrapolation. This poor documentation devalues much of the published information on the results of protein engineering. Nevertheless the specificity constant remains a meaningful concept in the two-substrate context. It has been used, for example, to describe the strikingly successful conversion of lactate dehydrogenase to malate dehydrogenase by a single mutation. In the mutant Q102R, derived from Bacillus stearothermophilus lactate dehydrogenase [5], the specificity constant for lactate decreased from 4.2 x 106 M- -l 1 to 5 x 102 M-1-.5 and that for oxaloacetate increased from 4 x 103 M-1 S-1 to 4.2 x 106 M-1. s-1 The same group of workers have now addressed the specificity of their enzyme for its second substrate, NADI, and attempted to engineer improved selectivity for NADP+ [6]. In presenting their results they employ a new parameter, which they term the 'overall catalytic efficiency', given by
substrate must be considered ........A realistic measure of overall catalytic efficiency must take account of the maximal turnover rate and Km values for both substrate and coenzyme." It is puzzling, first of all, that these authors have not similarly addressed the possible modulation of apparent selectivity for lactate versus malate by the coenzyme concentration. However it is not at all clear that the proposed new parameter is either needed or valid. It is indeed true that the apparent selectivity for either substrate is likely to be affected by the concentration of the other, but if there is concern for the way in which coenzyme selectivity might be affected by substrate concentration, it has to be met by reference to the rate behaviour at low concentrations of both substrates. As eqn. (1) shows, whereas qS is the proportionality constant with respect to [A] at high fixed [B], 5AB is the corresponding constant at low fixed [B]. The constant which Feeney et al. have introduced is
and this is only equal AB
to the constant 1/q5AB under the exceptional circumstances that the Km for A is independent of [B] and vice versa, i.e. the one set of circumstances where varying the fixed substrate does not affect the apparent specificity constant! This constant thus has no easily identifiable physical significance, and to describe it as a measure of overall catalytic efficiency is misleading. The original concept of a specificity constant, carefully applied,
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is still useful, but, as Feeney et al. [6] rightly point out, the mutual effects of the two substrates are of interest and importance. In order to excavate this information there is no alternative to carrying out systematic measurements that extend to low concentrations of both substrates simultaneously, so that the true 'combined specificity constant', 1/0AB, may be discovered. The Krebs Institute is a Centre for Molecular Recognition Studies under the Molecular Recognition Initiative of S.E.R.C.
Paul C. ENGEL Krebs Institute for Biomolecular Research, Department of Molecular Biology, University of Sheffield, P.O. Box 549, Firth Court, Western Bank, Sheffield SlO 2UH, U.K. 1. Fersht, A. R. (1985) Enzyme Structure and Mechanism, pp. 105-106, W. H. Freeman, New York 2. Cornish-Bowden, A. & Wharton, C. W. (1988) Enzyme Kinetics, p. 6, IRL Press, Oxford 3. Dalziel, K. (1957) Acta Chem. Scand. 11, 1706-1723 4. Engel, P. C. (1981) Enzyme Kinetics: the Steady State Approach (2nd edn.) Chapman & Hall, London 5. Wilks, H. M., Hart, K. W., Feeney, R., Dunn, C. R., Muirhead, H., Chia, W. N., Barstow, D. A., Atkinson, T., Clarke, A. R. & Holbrook, J. J. (1988) Science 242, 1541-1544 6. Feeney, R., Clarke, A. R. & Holbrook, J. J. (1990) Biochem. Biophys. Res. Commun. 166, 667-672 Received 29 January 1992
An improved assay for pyruvate dehydrogenase in liver and heart In a recent paper Paxton & Sievert [1] describe a new method to correct for the contribution of BCDH complex to the measured activity of PDH complex in tissue extracts. Others have used antibodies to BCDH [2]; or a kinetic method involving BCDH complex activity measured at saturating concentrations of ketoleucine, the Km for pyruvate of BCDH complex (734 gM), and the ratio of Vm.. values for ketoleucine/pyruvate of 5:1 [3]. The method in [2] is referred to in their paper; the method in [3] is not, though the reference is cited. The new method employs 14CO2 production from [1-14C]pyruvate to assay PDH complex activity and DCA and ketoisoleucine to inhibit 14CO2 production from pyruvate by BCDH complex. The method is potentially helpful but the purpose of this Letter is to draw attention to some ambiguities, misleading statements and unaddressed anomalies necessitating further consideration. The new assay uses 50,u/M-pyruvate according to the methods section (p. 547 paragraph 5), 0.5 mM-pyruvate according to the summary, the legend to Fig. 1 and Table 2 and text p. 548, paragraph 8. The concentration used in Table 1 is not stated. On p. 547, paragraph 2 it is stated that "the ratio of total BCODH to total PDC is about 0.65" and on p. 548, paragraph 8 the opposite is stated. Neither ratio is likely to be correct as the concentration of PDH complex is much too low. In liver mitochondria the ratio of total BCDH/total PDH is 0.2 on 17 % protein diet and 0.35 on 22 % protein diet [3,4]. Assuming that the new assay [1] uses 0.5 mM-pyruvate the contribution of BCDH complex to pyruvate decarboxylation (computed from kinetic constants above, [3]) is 8 % of BCDH complex activity measured at saturating [ketoleucine]. The highest estimates of total and expressed BCDH complex in liver
605 with ketoleucine are approximately 1.1 unit/g [4,8] giving a computed activity with 0.5 mM-pyruvate of 88 munits/g [expressed activity refers to the active (dephospho-) form of BCDH and PDH complexes and total activity to the sum of active and inactive forms. In livers of rats fed 22 % protein diets the two activities are virtually identical.] The total PDH complex activity of rat liver in [1] (Table 2) at 0.5 mM-pyruvate was 970 munits/g. Thus the computed contribution of BCDH complex to measured total PDH complex in Table 2 is 9 % and to expressed PDH complex activity (143 munits/g) is 61 % (a 6.8-fold difference). The values observed in Table 2 were 21 % and 27 % respectively. In an earlier paper reporting the antibody method [2] no effect of antibodies to BCDH on total PDH complex activity of liver was detected, even at 5 mM-pyruvate. There is no reference to or attempt to explain these apparent anomalies. In Table 2 of the paper by Paxton & Sievert [1] total PDH activities with 0.5 mM-pyruvate are given as 0.97 and 1.43 units/g (approximating to V.ax.) in livers of fed and starved rats respectively. These values are low for extracts prepared with 1 % Triton and protease inhibitors. Under comparable conditions of extraction (given in [4]) the value in this laboratory is 3.6 units/g fresh liver (fed) for total PDH with citrate synthase at 11 units/g (P. M. Stace & P. J. Randle, unpublished work). Walajtys-Rode & Williamson [5] measured 12.2 units PDH/g dry wt following extraction of hepatocytes with Lubrol (equivalent to 2.9 units/g wet wt liver). Table 2 of their paper also shows the effect of DCA/ ketoisoleucine to decrease 14CO2 production from [114C]pyruvate in liver. This decrement should presumably represent BCDH activity with pyruvate as substrate. The results show that the decrements for expressed and total BCDH activities were 33 and 169 munits/g respectively (fed) and 38 and 120 munits/g respectively (starved). It is well established that expressed and total activities of BCDH complex are essentially identical in livers of rats fed 22 % protein diets [4,8] (this diet was fed in [1]) i.e. the decrements for expressed and total activities should be the same. No explanation is offered for the 3-5 fold differences in the present study. If these unexplained discrepancies are due to errors inherent in the method then the method is clearly subject to substantial error and unacceptable. The data on rat heart in Table 2 shows even more striking discrepancies. The decrement in 14CO2 production from [1-_4C]pyruvate with DCA/ketoisoleucine was equivalent to 1 unit/g wet wt of BCDH complex measured with 0.5 mM-pyruvate as substrate for either total or expressed activity (fed rats). This is equivalent to 12.3 units BCDH complex/g wet wt with saturating ketoleucine (see paragraph 2 above). However measured total activities of BCDH complex in rat heart with ketoleucine as substrate are only 0.12 [19], 0.57 [20] and 0.3 units/g [21] with 48 % [19] or 9 % [20] or 18 % [21] of BCDH complex in the active form. These calculations indicate either that the decrement in 14CO2 production with DCA/ketoisoleucine is not due solely to inhibition of BCDH complex or that the errors inherent in the method are substantial. Table 2 in their paper also reports that 75 % of PDH is in the active form in hearts of fed rats, a result at variance with the 25-35 % found by others in hearts removed under pentobarbital anaesthesia. In paragraph 3 of Results and discussion the authors cite three references to work by others, which they claim shows that 60-80 % of PDH is active in hearts of fed rats. One of these [9] contains no measurements of % PDHa in heart; only measurements of % PDHa in heart mitochondria. Another [10] (from Dr. M. C. Sugden's laboratory) records a value of 59.4 %,
Abbreviations used: BCDH, branched chain 2-oxoacid dehydrogenase; PDH, pyruvate dehydrogenase; ketoleucine, 4-methyl-2-oxopentanoate; ketoisoleucine, 3-methyl-2-oxopentanoate; DCA, dichloroacetate. Vol. 284
