Project HL-16251 from the National. Institutes .... (type V from Clostridium perfringens) was pur- ... sodium sulfite (98%) from Matheson,. Coleman and Bell.
Vol.
252,
THE
JOLENAL
No.
7.2, issue
OF
BKILOG~CAL
of November Printed
in
L:
Reversible PROPERTIES
CHEM,WRY 25,
pp.
8035-8039,
1977
S.A.
Inactivation
OF THE
THREE
of L-Amino
CONFORMATIONAL
Acid Oxidase
FORMS* (Received
CHRISTOPHER
From
J. COLES,
the Molecular
Biology
94121 and the Department California
DALE
E. EDMONDSON,
AND
THOMAS
for publication,
June
17, 1977)
P. SINGER
Division, Veterans Administration Hospital, San Francisco, California of Bi0chemistr.y and Biophysics, University of California, San Francisco,
94143
L-Amino acid oxidase from snake venoms is known to undergo two types of reversible inactivation. One type is that induced by adjustment of pH to values near neutrality whereupon the enzyme spontaneously changes into an inactive configuration. On lowering the pH the active configuration is regained. In the pH range of 5.5 to 7.5, equilibrium states are reached where the more alkaline the pH, the more extensive is the inactivation. This type of inactivation process is prevented by monovalent anions, substrate, and substrate analogs and is characterized by a very high energy of activation. The enzyme is also inactivated progressively in the frozen state and is reactivated on heating at pH 5.0. This type of “freezing inactivation” is favored by acid pH and is not prevented by monovalent anions. Neither type of inactivation involves major changes in the physical properties of the enzyme. In the present study both the pa-induced inactivation and the freezing inactivation have been shown to cause substantial changes in the circular dichroism spectra of the enzyme in the 350 to 500 nm region, suggestive of conformation changes in the environment of the flavin. The circular dichroism spectra of the two inactivated forms differ from each other, however, as do the absorption spectra in the visible region. This is regarded as evidence that the two inactivated forms are not identical. Roth types of inactivation involve the loss of ability to bind the substrate, since the inactive forms are not bleached by L-leucine and do not bind anthranilic acid, a competitive inhibitor. Significant alteration of the electron affinity of the flavin coenzyme may also occur, as indicated by sulfite dissociation constants, which are higher by three orders of magnitude in the inactive forms than in the active enzyme. The energy of activation for the reactivation of both inactive forms is 45,300 + 1,300 cabmol, essentially the same value as has been determined for the pH-induced inactivation process. The latter process thus involves an entropic, rather than an enthalpic, change. Taken together, these observations suggest a conformational change in both types of inactivation occurring in the * This study was supported by Program Project HL-16251 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
region of the catalytic site of the enzyme and affecting both the flavin and the substrate binding region.
The reversible, pH-dependent inactivation of snake venom L-amino acid oxidase (EC 1.4.3.2) isolated in homogeneous form from Aghistrodon pisciuorus (1) and as the crystalline enzyme from Crotalus adamarrteus (21, was first described and extensively studied by Kearney and Singer (3-5) and more recently investigated by Wellner (6). It was shown (3) that the enzyme is spontaneously inactivated at pH values above 5.5. Inactivation progresses as a first order reaction and is characterized by a very high energy of activation (E,) of 42,500 cal mole-‘. On lowering the pH, activity returns completely. In the pH range of 5.5 to 7.5 an equilibrium exists between active and inactive forms, with a pK of 6.55, suggesting the role of a histidine residue in the interconversion (5). Inactivation is readily prevented by monovalent anions (41, substrates, competitive inhibitors, and riboflavin (51, but not by polyvalent anions, such as phosphate. Since no change in sedimentation velocity, electrophoretic mobility (51, or immunochemical response (6) was observed during the inactivation process, gross conformational changes, aggregation, or subunit dissociation may be ruled out as explanations of the phenomenon. No loss of the prosthetic group of the enzyme, FAD, occurs during the inactivation (5, 6). Small changes have been observed, however, in comparison of the absorption and optical rotatory dispersion spectra of the active and inactivated forms. These differences are most apparent in the region of flavin absorption, which is suggestive of a minor conformational change about the prosthetic group (6). This type of inactivation will be referred to as pH-induced inactivation in the present paper. Another unusual property of the enzyme is that it is inactivated on storage in the frozen state and is reactivated by incubation at 38” at suitable pH (1). Curti et al. (7) studied this phenomenon more extensively and reported that the rate of this type of inactivation is strongly dependent on the pH of storage and on the ionic composition, although, in contrast to the pH-induced inactivation, loss of activity in the frozen state is favored by low pH and is not prevented by chloride ions. No change in sedimentation constant, electrophoretic
8035
8036
Reversible
Inactivation
mobility, nor loss of FAD was observed on freezing inactivation. This process also appears to involve a limited conformational change affecting the environment of the bound FAD, since changes were noted in the visible absorption spectrum similar to those observed in pH-induced inactivation, together with a decreased rate of photoreduction of enzyme-bound FAD in the presence of EDTA (7). Some important questions have remained unanswered concerning both types of reversible inactivation. Are the forms of the enzyme produced in freezing inactivation and pH-induced inactivation identical? At what point in the catalytic cycle do these inactivation processes block the action of the enzyme? The present paper is addressed to these and related questions. EXPERIMENTAL
PROCEDURES
Materds and Enzyme Preparchons -L-[U-“CILeucine (specific radioactivity, 220 mCi/mmol) was obtained from the Biochemical and Nuclear Corp. Anthranilic acid (Aldrich) was decolorized with carbon and recrystallized first from toluene and then from water. Neuraminidase (type V from Clostridium perfringens) was purchased from the Sigma Chemical Co., Sephadex G-25 from Pharmacia, and anhydrous sodium sulfite (98%) from Matheson, Coleman and Bell. All other chemicals used were of analytical grade. L-Amino acid oxidase was isolated from C. adamanteus venom and twice recrystallized by the procedure of Wellner and Meister (2). The concentration of the enzyme catalytic sites (2/enzyme molecule) was determined from the FAD content (7). All enzyme concentrations stated in this paper refer to catalytic site concentrations. Assay Procedures-The activity of the enzyme was measured either polarographically (7) or spectrophotometrically by a modification of the procedure of Marcus and Feeley (8). In routine assays the reaction mixture (3 ml total volume) contained 67 rnM Tris/ chloride buffer, pH 7.8, 60 rnM KCl, 7 rnM L-leucine, 0.06 rnM 2,6dichlorophenolindophenol, and 1.08 rnM phenazine methosulfate. The dyes were added after equilibration at 38” and the reaction was started immediately by the addition of the enzyme. The reaction was followed at 600 nm and activity was calculated using the value E In\‘= 19.1 for 2,6-dichlorophenolindophenol. Under these conditions the measured activity was -3 times higher than in the polarographic assay, as expected from the fact that at the leucine concentration used here the rate of 0, uptake 1s nearly 3 times higher in 100% 0, than in air (1). The extrapolated maximal velocity from activity measurements at varying phenazine methosulfate concentrations (between 0.144 and 1.08 mM) for the rate of oxidation of 7 rnM Lleucine was -5 times higher t,han in the polarographic assay. Other Methods - The two types of enzyme inactivation discussed in this paper were carried out in 50 to 100 rnM sodium phosphate, pH 7.5 to 7.8, while activation was carried out in 200 rnM sodium acetate, pH 5.0. (Phosphate buffers were prepared without the addition of monovalent anions.) The enzyme was equilibrated with these buffers by passage through a column of Sephadex G-25 (1.25 x 26 cm) at O-4”. The temperature of incubation for pH-induced inactivation was 38”. Samples were periodically removed and assayed until the activity was 14% of the original and were used for experiments as soon as possible. Freezing inactivation was carried out in a commercial freezer set to -14”. Generally, >95% inactivation was obtained in 3 days. More than 90% of the original activity of samples inactivated by either procedure was restored by adjustment to pH 5.0 and warming at 38”. Absorption spectra were recorded at 15” with a Cary model 14 spectrophotometer interfaced to a Nova 214 computer (Data General Corp.) and circular dichroism spectra were measured at 15” with a Jasco ORD/UV5 spectrophotometer with Sproul Scientific SSlO modification. Sulfite affinity was determined by spectral titration (91, following the bleaching of the visible absorption of the enzyme-bound flavin, at 20 to 25 PM concentrations of enzyme. Spectra (600 to 300 nm) were recorded at 15” in 10 rnM sodium phosphate, pH 7.5. Sodium sulfite solutions were freshly made at 0” in the same buffer. Desialylation of the enzyme was achieved by incubation of the active form of the enzyme (1.4 mgiml, ‘-22 FM) with neuraminidase, 0.2 mgiml, at 37” for 24 h in 50 mM sodium acetate, pH 5.0 (10). A control, lacking neuraminidase, was similarly treated. The release
of
L-Amino
Acid
Oxidase
of sialic acid was determined on an aliquot with the thiobarbituric acid assay of Warren (11). The total sialic acid content of the enzyme was similarly measured after hydrolysis in 0.1 N sulfuric acid at 80” for 1 h (12). Neuraminidase was not removed from the samples for subsequent experiments. The substrate affinity of the enzyme was measured by equilibrium dialysis at O-4” in a microvolume dialyzer (Hoefer Scientific Instruments) under an argon atmosphere. Active and inactivated forms of the enzyme (prepared by pH-induced inactivation), at 18 FM concentration, were dialyzed against 100, 180, 350, and 600 PM LIU”C]leucine in 100 rnM chloride-free sodium phosphate, pH 7.8, using Visking dialysis membrane. Equilibrium was established after 48 h. Radioactivity was measured on aliquots mixed with Scintiverse (Fisher Scientific) with a Beckman model LS-150 liquid scintillation counter. The anaerobic generation of 2-keto-4-methylpentanoic acid from L-leucine was detected in the following manner. Active and inactivated enzyme, both 16 FM, in the buffer specified above, were mixed in anaerobic cuvettes under argon with LI U’*CJleucine (final concentration, 50 PM). Absorption spectra were measured before and after mixing. After a 15min incubation, Oifree trichloroacetic acid (final concentration, 5%, w/v) was added anaerobically to each sample. Concentrations of L-leucine and 2keto-4-methylpentanoic acid were determined by measuring the partition of radioactivity between equal volumes of 5% trichloroacetic acid and diethyl ether at 0”. Independent determinations showed that the amino acid partitioned 96% into the aqueous phase and the cu-keto acid 80% into the organic phase. Radioactive 2-keto-4-methylpentanoic acid was obtained from enzymatic oxidative deamination of L-[U’nClleucine and shown to be pure by thin layer chromatography (silica gel (Eastman-Kodak) developed with 1-butanoli acetic acid/water, 2:1:1, v/v) with an R, value of 0.82. Inhibition of activity by the competitive inhibitor anthranilic acid (13) was measured by the spectrophotometric assay in 100 rnM chloride-free sodium phosphate, pH 7.8, or in the Tris/HCl assay buffer described above. Assays were at 15” to prevent any inactivation while using the chloride-free buffer. L-Leucine concentrations were varied from 0.7 to 6 rnM and anthranilic acid concentrations from 0.3 to 1 mM. Inhibitor constants were determined from Lineweaver-Burk plots. The dissociation constant of the L-amino acid oxidase anthranilate complex was determined by spectral titration (13). The enzymes (active and both inactive forms) were 25 /*M. Spectra (730 to 330 nm) were recorded at 15” in 100 rnM sodium phosphate, pH 7.8. The increase in absorbance at 550 nm relative to 500 nm was used to calculate dissociation constants by the BenasiHildebrand method (14). RESULTS
Comparisons of Forms of Enzyme Obtained by Freezing studies Inactivation and pH-induced Inactivation ~ Previous on L-amino acid oxidase subjected to freezing inactivation (7) and to pH-induced inactivation (6) have shown that these processes cause qualitatively similar changes in the absorption spectrum of the enzyme-bound flavin. In order to evaluate whether the environment of the flavin is the same in the two inactivated forms, it was neressary to compare their absorption spectra under identical conditions. Fig. 1 shows that the spectra of the two inactivated forms of the enzyme in the visible and near-ultraviolet regions are clearly different from each other and from the active form. The spectral changes were fully reversible under the conditions of reactivation in both cases in agreement with previous results (6, 7). The difference spectrum observed on freezing inactivation (Fig. 1) is similar to that reported by Curti et al. (7) but shows larger absorption changes, possibly because of differences in temperatures and buffer composition in the two studies. The two inactivated forms of L-amino acid oxidase may also be distinguished by circular dichroism spectra in the spectral region where only electronic transitions ascribable to the flavin occur (Fig. 2). Since the isoalloxa+ne ring is optically inactive, the observed dichroic bands are due to the asymmetric environment of the protein binding site (15). Optical
Reversible
Inactivation
\
01
500
400
WAVELENGTH
of L-Amino
Acid
Oxidase
8037
-I
6cul
700
(nm)
FIG. 1. Visible absorption spectra of L-amino acid oxidase (25.2 in 55 rn~ chloride-free sodium phosphate, pH 7.5. -, active form; - - -, form obtained by pH-induced inactivation (active form maintained 15 min at pH 7.5 and 38”); ., freezing-inactivated form (active form maintained 3 days at -14”). The unset shows the difference spectra: - - -, form obtained by pH-induced inactivation minus active form; ‘, freezing-inactivated form minus active form. PM)
200
220 WAVELENGTH
240 (nm)
FIG. 3. Far-ultraviolet circular dichroism spectra of active enzyme (+--I, enzyme after pH-induced inactivation (- - -1, and by freezing inactivation ( 1. Conditions were as in Fig. 1.
-2.5[
I 350
I
8 450 WAVELENGTH
1 550
I
(nm)
FIG. 2. Visible and near-ultraviolet circular dichroism spectra of active form (-), form obtained by pH-induced inactivation (- - -1, and freezing-inactrvated (. ) form of L-amino acid oxidase in 10 rnM sodium phosphate, pH 7.5. The pH-induced inactivation was conducted as in the experiment of Fig. 1. Freezing-inactivated enzyme was generated from active enzyme by storage at -14” for 3 days in 100 rnM sodium phosphate, pH 7.8, followed by equilibration against 10 rnM sodium phosphate, pH 7.5.
activity induced through interaction of the flavin ring with the ribityl side chain and/or the adenine ring probably occurs also, although the spectra in Fig. 2 are clearly different from the published spectrum of FAD (16). The differences in circular dichroism spectra of the three forms of the enzyme shown in Fig. 2 reflect differences in the protein environment about the flavin. Circular dichroism spectral properties in the far-ultraviolet region, where dichroic bands due mainly to the n + n* and v --) v*. transitions of the peptide bond are observed, show little difference between the three forms of the enzyme (Fig. 3) in agreement with the optical rotatory dispersion data of Wellner (6). The polypeptide chain therefore does not undergo any major changes in conformation upon inactivation by either method. Some differences in the circular dichroism spectra of the inactivated forms were also noted in the 245 to 300 nm regions. These are not illustrated because of the difficulty of attributing these to a discrete component of the enzyme. Because of major differences in the conditions required for the two types of inactivation, comparison of the kinetics of the two processes is not practical, although they are known to proceed at very different rates (3, 7). The rates of reactivation
of the two inactive forms may be readily compared, however. This information is presented in the Arrhenius plot of Fig. 4. First order kinetics were observed throughout and least squares regression analyses were run on the data with the following results: following pH-induced inactivation, E,, for reactivation = 47,000 i 2,400 calimol; E,, for reactivation of the freezing-inactivated enzyme = 43,400 i 1,500 calimol, close to the published value (7). Thus, there is no significant difference between the E,, values for reactivation of the two inactivated forms. If the two sets of data are averaged, E,, for the reactivation process is 45,300 2 1,300 calimol. This value is represented by the line drawn in Fig. 4. The rate constants for the kinetic data above were found to be the same whether measured polarographically or spectrophotometrically. The E, value for the pH-induced inactivation in 55 mM phosphate buffer, pH 7.5, was found to be 42,100 -C 3,600 cali mol, in gratifying agreement with the value of 42,500 calimol reported over 25 years ago (3). Thus, the AH for the reversible pH-induced inactivation is essentially zero and the process involves an entropic rather t,han an enthalpic change. Characterization of Molecular Basis oflnactivation - While the flavin chromophore of active L-amino acid oxidase is rapidly bleached by substrate (1, 171, we find that both inactive forms of the enzyme are only slightly reduced by Lleucine anaerobically under similar conditions. The slight amount of reduction observed is rapid and corresponds to the residual small proportion of active enzyme. This level of reduction is unchanged after 45 min. This observation implied that the inactivated forms of the enzyme were either incapable of binding substrate or else that electron transfer between enzyme-bound substrate and the FAD could not take place. We attempted to distinguish between these possibilities by measuring the affinity of active and inactive forms of the enzyme for the substrate under anaerobic conditions. The experiment, using radioactively labeled L-leucine, as described under “Experimental Procedures,” was designed to detect the binding of radioactive
Reversible
8038
Inactivation
of L-Amino
Acid
Oxidase TABLE
I
Anaerobic generation of Z-keto4-methylpentanoic acid from Lleucine by active and inactive forms of L-amino acid oridase For conditions see “Experimental Procedures.” Equilibrium concentrations of components involved
in reaction
I*M
I
1
I
3.20
3.25 I/T
Active Inactiven None
I
330 x
lO-3
335
340
” Obtained
16 16 0 by pH-induced
22 3
28 41
0
50
inactivation
(K)
FIG. 4. Arrhenius plot for the reactivation of the two inactivated forms of L-amino acid oxidase in 200 rn~ sodium acetate, pH 5.0. Inactive forms of the enzyme were generated as in Fig. 1 prior to equilibration with the acetate buffer at 0”. Activity was monitored both polarographically and spectrophotometrically as described under “Experimental Procedures.” l , form obtained by pH-induced inactivation; X, form obtained by freezing inactivation.
TABLE
Dissociation
constants
complexes
of different
Conditions Enzyme
were
II
of anthranilic acid forms of L-amino
as under
“Experimental
complexes and sulfite acid ozidase at 15”
Procedures.” K,, values
form Anthranilic
acid
sulfite” mM
material up to a K,, of over 1 mrvr. No binding was observed to either active or inactive enzyme. Such weak substrate affinity was not unexpected for the inactive enzyme but was surprising for the active enzyme, since the K,, for the L-leucine has been reported as 1 mM (1, 17). It seemed likely that the active form had undergone one catalytic turnover, yielding the reduced flavoenzyme and a dissociable radioactive product (keto acid). This was confirmed by incubating both active and inactive Lamino acid oxidase anaerobically with a -3-fold molar excess of radioactive L-leucine and measuring the amounts of amino acid and n-keto acid at equilibrium. The results are presented in Table I. The active enzyme produced slightly more than 1 molar equivalent of Lu-keto acid, the excess most likely being due to residual oxygen, while the inactivated form generated less than 0.2 molar equivalent of ol-keto acid, which is due to traces of residual active enzyme and oxygen. These results suggested that the form of the enzyme obtained by pH-induced inactivation is incapable of binding L-leucine at the substrate binding site. Confirmatory evidence was obtained by studies with anthranilic acid, a competitive inhibitor of the enzyme (13). From steady state kinetic studies a K, value of 70 PM at 15 was obtained for the active conformation of the enzyme in 100 mM phosphate buffer, pH 7.8, under the conditions described under “Experimental Procedures.” At 15” the conversion of active to inactive enzyme is too slow to interfere with the experimental results. The dissociation constant of the enzyme. anthranilic acid complex was also measured by the spectral shift produced on binding the inhibitor (13). As shown in Table II, the dissociation constant determined at 15” in the same buffer was 68 pM in close agreement with the Ki value, but only one-sixth of that reported by DeKok and Veeger (13) at 25”. The discrepancy is not likely to be due to lo” difference in temperature, but to the high chloride concentration used by these workers. Monovalent anions are known to bind at or near the active site (4) and might be expected to interfere with the binding of competitive inhibitors. This was verified by ascertaining that the substitution of the Trisi chloride buffer used by DeKok and Veeger for phosphate resulted in a severalfold increase in the K, value. The differences in K, and K,, values for anthranilic acid reported by these workers are also almost certainly due to their use of
Active pH-induced activated Freeze-inactivated ,, K
0.068
in-
>lOb
0.042 68
>lOb
63
= ~nzyme:FADl[sulfite + bisulfitel [enzyme:FAD - sulfite complex] b No spectral changes observed up to 1 rnM anthranilic IJ
acid.
different chloride concentrations in the two determinations. Table II demonstrates that neither type of inactivated enzyme binds anthranilic acid, although binding would have been detected up to a K, value of 10 mM. These results support the conclusion that the reversibly inactivated forms lack the requisite conformation for normal binding of substrates and substrate analogs. The differences between the active and inactive forms of the enzyme also include the properties of the flavin, as is evident from the data in Figs. 1 to 3. Preliminary studies’ have indicated that large changes in the oxidation-reduction potential of the enzyme-bound FAD take place on inactivation of the enzyme. It was appropriate, therefore, to measure t,he reactivity of different forms of the enzyme with sulfite (9) since sulfite affinities and oxidation-reduction potentials are interrelated in free flavins (18) and probably also in flavoenzymes. The K,, obtained in chloride-free buffers for the active enzyme (Table II) is slightly lower than the published value (9), which was determined in the presence of chloride. More importantly, both inactive forms show drastically reduced affinity for sulfite, the K,, values being 3 orders of magnitude greater, at just over 60 mM, which is consistent with a lowering of oxidation-reduction potential (18). As with Damino acid oxidase (91, the reversible nature of the sulfite complex with the active enzyme was demonstrated by the addition of a competitive inhibitor (anthranilic acid), which slowly caused reversal of the bleaching by sulfite. L-Amino acid oxidase is a glycoprotein, containing about 1 mol of sialic acid/m01 of FAD (191, a value confirmed in the present study. The carbohydrate portion of the enzyme does not seem to be involved in the conformation changes related ’ D. E. Edmondson
and C. J. Coles,
unpublished
results
Reversible
Inactivation
to the activation-inactivation cycle, since removal of over 90% of the sialic acid from the active enzyme with neuraminidase caused no change in activity or in the rate and extent of inactivation and reactivation. DISCUSSION
Previous studies by Kearney and Singer (3) and Wellner (6) on the pa-induced inactivation of L-amino acid oxidase and by Curti et al. (7) on the freezing inactivation have disclosed several similarities between the two processes. Thus, both appear to involve very limited conformational changes, since a variety of physical methods and immunochemical techniques fail to distinguish between the active and reversibly inactivated forms. This conformational change appears to cause a hypsochromic shift in the visible absorption spectrum of the enzyme in both cases (6, 7). Further, the same conditions bring about reactivation after both types of inactivation (3, 7). From the data presented above, it is clear that the energy of activation for the reactivation process is also identical and that both processes involve loss of the ability to bind substrate and competitive inhibitors and lowers affinity for sulfite. The latter suggests a major decline in the oxidationreduction potential on inactivation. Despite the similarities, comparison of the absorption (Fig. 1) and circular dichroism spectra (Fig. 2) under identical conditions reveals differences between the two inactive forms. Differences in the effects of pH and anions on the two types of inactivation have been previously pointed out (7). It may be concluded, therefore, that L-amino acid oxidase exists in at least three relatively stable conformations: the active enzyme, the freezing-inactivated form, and the form resulting from pH-induced inactivation. A fourth stable conformation of the enzyme, designated the “y” form, has been studied by Wellner and Hayes (20). This inactive form of the enzyme arises from incubation of the pH-induced inactivated form of the enzyme with p-chloromercuribenzoate at pH 5.0 at 0” for several hours. It remains for future work to determine if the freezinginactivated form of the enzyme can be converted to the “7” form on treatment with p-chloromercuribenzoate. The present study permits a somewhat closer delineation of the reasons for the lack of catalytic activity of the two reversibly inactivated forms than has been hitherto possible. Both inactivations affect the flavin, as well as the substratebinding region of the catalytic site. The effects on the flavin site are evident from the shifts in the absorption spectrum (6, 71, from the altered circular dichroism spectra (Fig. 2), and from the lowered affinity for sulfite (Table II). The fact that the substrate site may also be altered was first suggested by the observation that L-leucine bleaches the active but neither type of inactive form. This experiment indicated either that the two inactive forms cannot bind the substrate, or that if they do bind it, reduction of the FAD moiety by the enzyme-
of L-Amino
Acid
8039
Oxidase
bound substrate is blocked. Evidence for the first alternative came from the demonstration that the inactive forms do not bind the competitive inhibitor anthranilic acid to any significant extent (Table II). The same conclusion is reached from the findings that neither binding of L-[‘Clleucine by the two inactive forms is evident nor conversion of leucine to 2-keto-4methylpentanoic acid (Table I). (Although the binding of L[“Clleucine also has not been demonstrable with the fully active enzyme on anaerobiosis, in that instance, a nearly stoichiometric amount of the a-keto acid is formed, showing that the binding of the substrate, oxidation of the substrate by the flavin, and release of the product had occurred.) The lack of catalytic activity by both inactivated forms of the enzyme is thus ascribable to their inability to bind substrates. Whether these forms would be able to catalyze electron transfer between the bound FAD and the substrate if the latter could be bound is a moot question in view of the altered properties of the flavin, particularly in view of the likelihood that a major decline in its oxidation-reduction potential occurs on inactivation.’
Acknowledgment-We the
wish dichroism
use of his circular
to thank Dr. spectrometer.
J. T.
Yang
for
REFERENCES 1. Singer, phys. 2. Wellner,
2018 3. Kearney, phys.
T. P., and Kearney, 29,
E. B. (1950) Arch.
Biochem.
Bio-
190-209
D., and Meister, E. B., and
A. (1960)
Singer,
J. Biol.
T. P. (1951) Arch.
Chem.
235, 2013-
Bmchem.
Bio-
33, 377-396
4. Kearney, E. B., and Singer, T. P. (1951) Arch. Biochem. BLOphys. 33, 397-413 5. Kearney, E. B., and Singer, T. P. (1951) Arch. Bzochem. Biophys. 33, 414-426 6. Wellner, D. (1966) Biochemistry 5, 1585-1591 7. Curti, B., Massey, V., and Zmudka, M. (1968) J. Biol. Chem. 243, 2306-2314 8. Marcus, A., and Feeley, J. (1962) Bmchrm. Biophys. Acta 59, 398-407 9. Massey, V., Miiller, F., Feldberg, R., Schuman, M., Sullivan,
10. 11. 12. 13.
P. A., Howell, L. G., Mayhew, S. G., Mathews, R. G., and Foust, G. P. (1969) J. Biol. Chem. 244, 3999-4006 Cassidy, J. T., Jourdian, G. W., and Roseman, S. (1965) J. Biol. Chem. 240, 3501-3506 Warren, L. (1959) J. Biol. Spiro, R. G. (1966) Methods
DeKok,
A., and Veeaer,
Chem. 234. 1971-1975 Enzymol. 8, 3-26 C. (1968) Biochm. Bzophys. __
Acta
167,
35-47 14.
Benasi, H. A., and Hildebrand, J. H. (1949) J. Am. C&m. Sot. 71, 2703-2707 15. Edmondson, D. E., and Tollin, G. (1971) Bzochemistry 10, 113123 16. Miles, D. W., and Urry, D. W. (1968) Biochemistry 7, 2791-2799 17. 18. 19. 20.
Massey,
Miiller, DeKok, Wellner, 151,
V.,
and
Curti,
B. (1967)
J. Bzol.
Chem.
242,
1259-1264
F., and Massey, V. (1969) J. Biol. Chem. 244, 4007-4016 A., and Rawitch, A. B. (1969)Biochemistry 8, 1405-1411 D., and Hayes, M. B. (1968) Ann. N. Y. Acad. SCL. 118-132
