THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol.246, No. 24,Issue of December 25,~~. 76667674,1971

Printed in U.S.A.

Binding Site Determination from Kinetic Data

REDUCED E’LAVIN MONOIWCLEOTIDE BINDING TO BACTERIAL LUClI’ERASE”

(Received for publication, June 14, 1971)

EDWARD A. MEIGHEN!: AND J. WOODLAND HASTINGS~

Prom The Biological Laboratories, Harvard University, Calnb&ge, Massachusrifs ~l,%l,%

SUMMARY

The elucidation of the number of reduced flavin mononu- cleotide (FMNHt)-binding sites in bacterial luciferase is of fundamental importance for understanding the mechanism of the bioluminescent reaction. At present, knowledge con- cerning the binding sites is meager, primarily due to difhculty in maintaining FMNHt in a reduced state at very low concen- trations. In the present studies this difficulty has been circumvented, permitting us to elucidate the order of the bioluminescent reaction with respect to FMNHs and the dis- sociation constant of the FMNH2-enzyme complex. Of par- ticular importance was the fact that enzymatic measurements could be made not only at extremely low FMNH2 concentra- tions (lOug M), but also at very high enzyme concentrations, even higher than the substrate concentration or the value for the dissociation constant for the FMNH2-enzyme complex (8 x low7 M). Under these conditions, the Michaelis- Menten equation is not directly applicable and the relative velocity of the reaction (u/V,,,) depends on the concentra- tion of substrate-binding sites. Consequently, it was possi- ble to elucidate the number of FMNH2-binding sites directly from kinetic data. These results show that bacterial lucifer- ase possesses only a single FMNH,-binding site and con- sequently eliminate reaction mechanisms involving 2 FMNHz molecules. Since the oxidation of a single FMNHz molecule would not be adequate energetically for the emission of a quantum of light at 490 nm, additional energy-yielding steps must be required in the bacterial bioluminescent reac- tion.

Although bacterial luciferase has been extensively studied, knowledge concerning the binding sites for the substrate, FMNHz, is meager. This is due in large measure to technical difficulties involved in its assay, principally to the fact that the nonenzymatic autoxidation of FMNH2 by molecular oxygen is rapid and competit.ive wit’h the cnzymat’ic pathway. In the

* This work was supported in part by National Science Founda- tion Research Grant GB 16512.

$ Present address, Department of Biochemistry, hZcGil1 Univer- sity, Montreal, Quebec, Canada.

0 To \?-hem reprint requests should be addressed.

present study these difficult,ies have been studied and partially circumvented, allowing us to describe kinetic aspects of the reac- tion in detail. Not only has the dissociation constant of the FMNHz-enzyme complexbeen elucidated; the number of binding sites has also been determined directly from kinetic measure- ments.

The bioluminescent oxidation of FMNHz cat,alyzed by bac- terial luciferase is unusual in several respects (1). The reaction is highly exergonic, involving a large AF”, one of the products being a photon (490 nm) equivalent to about 60 kcal per Einstein. The reaction kinetics is even more unusual. The nonenzymatic autoxidation of free FMNH2 occurs within less than a second (k = 10 see-l, 25”) (2), whereas the enzymatic reaction takes 10 to 100 times as long. The practical consequence is that the classical enzyme-substrate complex has a long lifetime, measured in seconds or minutes, depcndin g on experimental condit8ions. From this it follows that the enzyme functions (turns over) only once in an in vitro assay whcrc reduced flavin and enzyme are mixed in the presence of oxygen. fl final unusual feature relates to the requirement for a long chain ( > 7 carbon atoms) aliphatic aldehyde: the over-all enzymatic reaction, involving the oxida- tion of FMNI-12, occurs I\-ilh similar (although not idemical) kinetics with or without :tldchytle, but the photon gicld is signif+ cant only in its presence (3, 4).

From prior studies a react,ion scheme (Fig. 1) was hypothesized (3) in which the reart#ion of the enzyme with reduced flavin re- sulted in the formnt’ion of an intermediate (I) whose reaction with oxygen produced a long lived intermediate (II). The In- termediates I and II are treated as enzyme-substrate complexes, ES and ES”, in the present paper. In the scheme proposed the further reaction of Intermcdiatc II in the presence of aldehyde gives light with a high quantum yield; without aldehydc a further reaction occurs at a sirnilar rate but with a lOOO-fold lower quan- tum yield. The excited species are both arbitrarily designated as (E-F) *, and may or may not be identical in the two cases.

Because of the unusual features of the reaction, especially the long lifetime of the intermediate, and also because the formation of product (photon emission) cau be detected at unusually low levels (many orders of magnitude below t,hat availablo by con- ventional techniques), measurrment,s can be made in concentra- tion ranges not normally nc~ces&lc. Enzymatic activit’ies (ini- tial velocities) can be accurately measured at conccntrat;ions of FMNHz as low as 10-g x Determinations can also hc made at very high luciferase concclltrations, both greater than the sub- strate concentration and greater than the value for the dissocia-

7666

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Issue of December 25, 1971 E. A. Meighen and J. W. Hastings 7667

tion constant of the FMNHz-enzyme complex. These measure- ments permitted us to elucidate the number of FMNHZ-binding &es per luciferase molecule in a unique way, directly from kinetic measurements.

EXPERIME?iTAL PROCEUURk!

Materials-Bacteria1 luciferases were isolated from Photobac- teriunz fischeri, strains MAV and Pf No. 7744, and purified ac- cording to the procedure of Gunsalus-Miguel et al. (5). The purified proteins, MAV and Pf luciferases, had specific activities of 1,000 and 20,000 light units per mg assayed at 22” with dodcc- anal. One light unit is equivalent to 2.2 x 1O1” quanta per sec. The protein concentrations were determined spectrophoto- metrically on the basis of a specific absorbance coefficient of 0.94 (O.l%, 1 cm) at 280 nm. A molecular weight of 7.9 X lo4 was used in all calculations (6). Unless otherwise specified, the MAV luciferase was used.

FMN was a gift of Sigma and was used directly. Its concen- tration was based on a molar extinction coefficient of 12,200 (1 cm, 450 nm) (7) and may be slightly high due to impurities. Phosphate buffers were made by mixing appropriate amounts of KzHPOI and NaH2P04. All other chemicals were of reagent grade.

Standard Assay-The standard in vitro assay for luciferase ac- tivity was carried out by the rapid injection of 1 ml of 5 x 1O-5 M

FMNI-12 (catalytically reduced with Hz over platinized asbestos) into a reaction mixture (1.2 ml) containing luciferase, aldehyde, and oxygen in 0.2 y0 BSA,’ 0.02 RI phosphate at pH 7. The alde- hyde (decanal or dodecanal) was added to the assay mix as a 0.1 y0 (v/v) sonicated suspension, with approximately 0.015 and 0.1 ml for MAV and Pf luciferase, respectively.

Dithionite Assay-FMN in a volume of 1.0 ml was added to luciferase in 1.2 ml of 0.02 M phosphate, 0.2% BSA, pH 7.0. With a spatula, excess solid sodium dithionite (Na&OJ was added so that no FMN fluorescence could be observed. The reaction (with MAV luciferase) was initiated by the rapid injec- tion of 1.0 ml of a 0.005% (v/v) sonicated suspension of aldehyde (decanal or dodecanal) from a hypodermic syringe. For Pf luciferase, 1.0 ml of a 0.1 y0 aldehyde suspension was injected. Constant activities were obtained for variable amounts of Naz- SZ04 (between 0.2 and 1.0 mg); no luminescence was obtained if too little NazSz04 was added. In such instances the fluorescence of oxidized flavin was already apparent before injection of alde- hyde (02). I f an excess of NazSz04 was added, then lower light intensities were obtained, presumably because the quantity of 0% in the aldehyde suspension was not adequate to saturate the enzyme before the oxygen was reduced by the excess Na8204. There were instances, however, in which it was noted that flavin was still reduced (no fluorescence) after the completion of the reaction, and yet full activity was apparently obtained. This result shows first that the enzyme-FMNHt complex can very effectively compete with the excess Na&&04 and free FMNHz for the O2 molecules in the aldehyde suspension, and secondly that free oxygen is not necessary for the persistence and ultimate emission from the long lived intermediate (II). Indeed, the in- termediate is stable t,o reducing agents, a fact which was noted some years ago (3).

Unless otherwise noted, the bioluminescent assays were con- ducted at a constant temperature (~0.5’) between 22 and 25”.

1 The abbreviation used is: BSA, bovine serum albumin.

E + FMNH2 non-enzymatic

02 l FMN + H202

I E-FMNH2 (II

02 4

E-FMNH2-O2 (II)

old - E - aldehyde

(E-F)” (E-F)”

I high quantum

I

low quantum yield yield

hv hv

FIG. 1. Reaction scheme for the bioluminescent oxidation of reduced flavin mononucleotitle.

The FMN concentration used was that calculated prior to injcc- tion of the aldehyde suspension. Light emission was detected by the use of a photometer (8) and recorded graphically.

RESULTS

Steps in Binding of FNNII~ to Luciferase-The bacterial bio- luminescence reaction may be initiated in vitro by the rapid mix- ing of FMNHz with luciferase, the latter in a solution containing a long chain aliphatic aldehydc and oxygen. After initiation of the reaction, the light intensity rises rapidly to a maximum and then decays exponentially (Fig. 2). In addition to its reac- tion with luciferase, the reduced FMN is rapidly autoxidized (2) (nonenzymatically) (t+ = 0.1 set). However, since light persists for a considerably longer period (t+ decay = 2 to 100 EC), a stable intermediate must be produced in the bioluminescent reac- tion whose decay results in the emission of light. In this assay the enzyme can thus function (turn over) only once with a single injection of substrate.

The steps in the bioluminescent reactlion (see the introduction) at saturating concentrations of O3 and long chain aldehyde may be represented as shown below

h E+S ---‘ESLEX” h

---+ light (hv) k-1

where FMNHs is designated by S, and ES and ES” refer t#o In- termediates I and II, respectively. The number of moles bound and the order of the bioluminescent reaction with respect to FMNIlz will be considered later. The reduced flavin enzyme complex (ES), formed at a rate given by kl, reacts with oxygen to form an intermediate (ES”) with a pseudo-first order rate con- stant, kz. The intermediate (ES’) is extremely stable, with a lifetime of seconds, and is slowly converted to product with light emission, at a rate given by kl. The initial light intensity (lo) and the related chemiluminesccnt quantum yield, are propor- tional to the amount of this intermediate (9). In addition, the

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7665 Determination of FMNHS-binding Sites on Bacterial Luciferase Vol. 246, No. 24

4 8

Time (set> FIG. 2. Effect of initiation method on light intensity in the in

vitro bioluminescent reaction. In all cases, the assay mixture con- tained identical amounts of MAV luciferase (2 rg) in 0.2% BSA, 0.02 1\1 phosphate, pH 7.0. The ordinate gives the light intensity in light units (LU) on a logarithmic scale and the abscissa the time after initiation of the reaction. d, standard assay. Injec- tion of 1.0 ml of catalytically reduced FMNH, (5 X 10e6 M) into 1.2 ml of the assay mixture containing 15 ~1 of 0.1% (v/v) sonicated suspension of decanal (X). B, injection of 1.0 ml of FMN (5 X 10-6 M) and Na&O, (~0.5 mg) into I.2 ml of assay mixture con- taining 15 ~1 of 0.1% (v/v) sonicat,ed decanal suspension. The Na&04 was added as a solid powder to the FMN solution just prior to injection. The final assay solution could either be re- duced (m) or oxidized (0). C, dithionite assay. Injection of 1.0 ml of 0.005% (v/v) suspension of decanal into 2.2 ml containing 1.2 ml of assay mixture, 1.0 ml of 5 X 10-b M FMN, and freshly added Na&Oh (~0.5 mg). The final assay solution could either be reduced (0) or oxidized (0). D, injection of 1.0 ml of cata- lytically reduced FMNHt into 1.2 ml of assay mixture containing 15 ,~l of 0.1% (v/v) sonicated decanal suspension and oxidized Na&04 (~0.5 mg) (A). The Na&Od was oxidized in the assay mixture prior to the aldehyde addition and the subsequent FMNH2 injection.

above reaction scheme is complicated further by the rapid autoxi- dation of FMNHz (8)

FMNHz h

+a + FMN + Hz02

where the pseudo-first order rate constant, kq, is much greater than ka.

Initiation of Reaction: E$ect of Dithionite and Order of Substrate Addition-Significant differences in the light output (i.e. reaction rate) were noted when the bioluminescent reaction was initiated by different methods (Fig. 2). In the commonly used assay FMN is reduced catalytically and injected at saturating concen- trations from a syringe into the assay solution containing lucif- erase, aldehyde, and oxygen (Curve A). I f the assay is carried out identically except that the fiavin is reduced by dithionite (NazSz04), the light output is far lower (-15%, Curve B). But if both the luciferase and the same amount of flavin are reduced together with dit,hionite, and the reaction initiated by injecting

an air (O&equilibrated aldehyde solution, the light output (Curve C) is the same as in the first (standard) assay. In both assay systems, B and C, the final assay solution could be either reduced or oxidized without greatly affecting the relative light intensities. Addition of a larger excess of NaS204 (> 1.0 mg), however, reduced the light intensities in both cases. Experi- ments (Assay D) in which Na2S204 was oxidized in 1.2 ml of the standard assay solution and then aldehyde and luciferase added followed by the rapid injection of 1.0 ml of catalytically reduced FMNHZ gave the same result as Assay B.

The explanation for the differences appears to be due to the following circumstances. Dithionite, or its oxidation products, or both, greatly accelerates the oxidation of FMNHz (k4 = 100 see-1 (2)). Thus in the experiment of Curve A the luciferase is saturated with FMNHz at the concentration used, whereas in Assays B and D, saturation is not achieved because of the more rapid autoxidation of FMNH2. Assay D indicates that the oxidation products of dithionite rather than dithionite itself accelerate the oxidation of FMNH2. The fact that FMNHs is not satura,ting may be shown by increasing the FMNHz concen- tration. In Curves A and C no further light output is obtained; in Curve B the light emission (IO) is increased about 3-fold when a lo-fold higher substrate concentration (5 X 1O-4 M) is injected.

Two reasons for Assays B and D not yielding maximum ac- tivity may be considered. First, the rate of FMNHz binding to luciferase may be slow relative to the rate of FMNHz oxidation. Alternatively, it may be that the FMNHs binding is rapid but the subsequent interaction of oxygen with the FMNHz-enzyme complex (ES) is relatively slow. In the first case, not all of the enzyme molecules could interact with FMNHt prior to its autoxi- dation to FMN, while in the other case, not all of the ES mole- cules could interact with O2 prior to the autoxidation of FMNHz and the reversion of ES to reactants. A higher concentration of FMNI-12 would result in greater activity in both cases. In the first case, more of the enzyme molecules could be converted to EX (see Equation 7a), while in the latter case, the ES molecules would be available for interaction with 02 for a longer period of time (see Equation 11). However, preincubation of FMNHs with luciferase (Assay C) should result in greater light production in the former case, but have little or no effect on activity where FMNHz binding is relatively rapid. Since Assay C gives maximum light production, we conclude that the lower activity observed in Assay B results from the slow rate of formation of ES. This also requires that the rate of dissociation of the FMNHz-enzyme complex (L1) must be less than its rate of reaction with O2 (kJ.2 Otherwise, we would not observe the complete conversion of E to ES in Assay C and only 1501, con- version to products in Assay B.

Previous experiments by McElroy and Green (10) investigated the relationship between light intensity and FMNHz concentra- tion by the standard assay procedure (A) given in Fig. 2. These experiments gave an approximately linear relationship between l/v and 1/x2, thus indicating the bioluminescent reaction was second order with respect to FMNHz concentration. In a rein- vestigation of this relationship we have found a high variability

2 Strictly speaking, this conclusion does not necessarily apply in solutions in which NazSz04 is absent (i.e. the standard assay procedure). In the cases upon which this conclusion was based, Na&O, was present, and it (or its reaction products, or both) may have caused an increase in the reaction rate of the FMNHz-enzyme complex with 02.

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Issue of December 25, 1971 E. A. Meighen and J. W. Hastings 7669

in the reaction velocity (13 in experiments using low FMNH2 concentrations. In these assays, FMNHz is obtained by bub- bling Hz through an FMN solution containing platinized asbestos. The FlMNHz is then withdrawn in a syringe and rapidly injected into the assay solution. Much higher light intensities were gen- erally obtained if the time between withdrawal and injection of the FMNHz was short (approximately 2 set) rather than long (approximately 10 set). Apparently, the FMNHz was being autoxidized in the syringe prior to injection into the assay, the effect being more significant at low FMNHz concentrations. These results indicated that it would be relatively difficult to investigate quantitativeiy the binding of FMNHZ to luciferase by this assay.

However, such problems would not be encountered by assays in which excess NazSz04 is added to maintain the FMNHz in the completely reduced state prior to mixing in the assay solution with OZ. The injection of the aldehyde suspension as in Assay C (dithionite assay) is thus more suitable for the investigation of the binding of FMNH?.

Relationship between Light Intensity and FMNH2 Concentration -Since the dissociation of FMNHz from luciferase (L1) is slow relative to the reaction of 02, both with ES (kt) and with the substrate (k4), the injection of the oxygen-aldehyde suspen- sion (Assay C) into a system in which the equilibrium

h ESS - ES

Ic_t

is established should quantitatively convert ES to ES”, and the excess (free) FMNHz (8) to FMN. Thus the amount of ES” formed in this assay is simply given by

(ES”) = (ES) = m d

where Kd is defined as the dissociation constant (Ll/kJ for the FMNHZ-enzyme complex, and (ET) is the total enzyme concen- tration ((E) + (ES)).

As the initial light intensity is proportional to the amount of stable intermediate (ES”), we can write Equation 1 in the form of the Michaelis-Menten equation (11)

v max 69) v=Ka

(2)

where v is the initial light intensity (lo) and V,,, is the initial light intensity at saturating FMNHz concentrations. If the order of the bioluminescent reaction (n) with respect to FlMNHe is also considered, Equation 2 becomes

vmx Lw ’ = Kd + (5’)”

(3)

Rearranging and taking the logarithm gives the following rela- tionship

iog(~-I)=loi:Kd-nio::(S) (4)

for assays in which FMNHz and enzyme are preincubated to- gether.

For the assays involving injection of FMNHZ (Fig. 2, B and D), the relationship between light intensity and FMNHz con- centration is more complicated than the assay described above.

In these assays the conversion of E to ES (product) would be given by

where (Xi) and (Et) vary as a function of time, t, after inject,ion of FMNH2, and kr is the second order rate constant for binding of FMNHS to luciferase. Since the reaction of oxygen with the FMNHz-enzyme complex (kz) is more rapid than its dissociation, this reverse reaction (k-J does not need to be considered. The FMNHz concentration at any time, t, is given by

(S,) = (S)e-% (6)

where (8) is the initial FMNHS concentration and k4 is the pseudo-first order rate constant for nonenzymatic autoxidation of FMNHz. Substituting into Equation 5 and integrating be- tween t = 0 and t = w gives

(E”) = (Ep)e-kl(S)lk4 (7)

where (E”) is the free enzyme that has not turned over in the reaction. Since the total amount of ES” formed is given by (ET) - (E”) we can now write

(ES”) = (ET)(l - e-kl(s)/kl) ?a)

If we consider the order of the bioluminescent reaction (n)

with respect to FMNH*, Equation 7a becomes

v = v,,, (1 - e--kl(S)‘k4n) (8)

where v/Vm,, = (ES”) /(ET). A comparison of the above Equa- tion 8 with Equation 3 shows that entirely different relationships exist between the initial light intensity (v) and substrate concen- tration (8) for the two different methods of initiation of the bioluminescent reaction.

In contrast, if the equilibrium between FMNH2 and enzyme had been rapid compared to the interaction of ES and FhIN& with oxygen then for both assay methods (injection and pre- liminary incubation of FMNHz), the stable intermediate would have been formed at a rate given by

c&W,“) - = ME&) clt

where kt is the pseudo-first order rate constant for interaction of the FMNHz-enzyme complex with oxygen. The extent of con- version of ES” to light during its formation from reactants can be considered to be negligible, as kz is much greater than ka. Since ESt would, by definition, be in rapid equilibrium with free enzyme and substrate under these conditions, it follows that

[(ET) - (ES,“)](&) mu = - Ic d + (s )

t (10)

Substituting Equations 6 and 10 into Equation 9 and integrating between t = 0 and t = m gives

b%S’“) = (ET) [I - (&)Llik4] (11)

If the order of the bioluminescent reaction with respect to Fh4NH2 is also considered then Equation 11 would be written as

v = v,,, [l - (K*n) ““Q] (12)

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7670 Determination of FMNH2-binding Sites on Bacterial Lucijerase Vol. 246, No. 24

where v/V,,~ = (&Y’)/(Er). This equation would apply to both methods of assay (either preincubation or injection with FMNH2) if the interaction of oxygen with FMNHz and the FMNHz-enzyme complex had been slow compared to the rate of

FIG. 3. A plot of log (Vmax/v - 1) versus log S for MAV lucifer- ase. The initial light intensities (v) at different FMNHS concen- trations (moles per liter) were obtained by the dithionite assay as given under “Experimental Procedure.” The FMNHz concentra- tions (S) used in the above plot were those calculated prior to injection of the aldehyde (dodecanal) suspension. Luciferase con- centration was 8 X 1O-g M. The maximum light intensity (V,,,) was obtained at saturating FMNI& concentrations.

4

FIG. 4. Relationship between the reciprocal of the initial light intensity, (l/v) and the reciprocal of the FMNHz concentration in moles per liter (l/S) for MAV luciferase. Additional details are given in Fig. 3.

equilibration between FMNH2 and luciferase. From the ex- perimental results (see Fig. 2), it has been clearly shown that this is not the case.

F11fNH2 Binding to ilsA4J’ Lucijerase-The experimental rela- tionship between initial light intensity (v) and FMNHz concen- tration (8) was investigated for MAV luciferase by the dithionite assay (preincubation of enzyme with Fh!lNHz and Na&Od; see “Experimental Procedure”). The results are plotted in Fig. 3 according to Equation 4 and give a linear relationship between log (Bma& - 1) and log S over a lOOO-fold substrate concentration range, with a slope equal to -1.0. This result shows that the experimental data are described by Equation 2 and that the bioluminescent reaction has a first order dependence on the FMNH? concentration.

The experimental data are plotted in Fig. 4 according to a Lineweaver and Burk (12) plot and give a dissociation constant (KJ for FMNHz equal to 8.0 x 1OP 31. The Rd was not affected by the chain length of aldehyde used in the assay (dec- anal or dodecanal) or the enzyme concentration (where (ET) << Kd .

Number of FJlNH2-binding Sites in MAV Lucijerase-The above experiments on MAV luciferase were conducted at enzyme concentrations well below the Kd for FMNH2. Since the en- zymatic activity of luciferase can easily be measured over a con- centration range of 107, assays can be conducted at protein con- centrations ranging from 3 x 1O-12 to 3 x lop5 M. Thus, binding experiments can be conducted with luciferase at enzyme concen-

4or

---A- .-.I ..-.- 12

‘/s x IO-+ FIG. 5. A plot of ‘Vmlna/v versus 1/X at different MAV luciferase

concent,rations (8 X 10eg M (0); 1.04 X 1OW M (0); 2.08 X 1W6 M (A)). The limiting slope at low substrate concentrations gives the sum of the concentration of FMNHZ-binding sites and the value for the dissociation constant of the FMNHZ-enzyme com- plex (& + E) in moles per liter.

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Issue of December 25, 1971 E. A. Meighen and J. W. Hastings 7671

FIG. 6. A plot of S (Vmnr/v - 1) versus (1 - v/V,,,) at different MAV luciferase concentrations (8 X lO+ M (0) ; 1.04 X 1OW M (0) ;

2.08 X 1O-6 M (A)). The experimental data at low enzyme con- centrations (0) were fitted with a horizontal line with the mini- mum standard deviation. The data at high enzyme concentra- tions (A and 0) were fitted with straight lines intercepting the ordinate at a common point with the minimum s&ndard deviation. The slope gives the concentration (M) of FMNHZ-binding sites and the intercept the dissociation constant for the E’MNHz-enzyme complex.

* 0 E, .A.---I -I

2 3

I/(1-“/v,’ FIG. 7. A plot of S (V,,,/v) uersus l/(1 - v/Vmax) at different

MAV luciferase concentrations (8 X 10e9 M (0 ) ; 1.04 X 10e6 M (0) ; 2.08 X 1OW M (A)). The experimental data at low enzyme concen- tration ( l ) were fitted with a line that intercepted the origin with the minimum standard deviation. The data at high enzyme con- centrations (A and 0) were fitted with straight lines with the minimum deviation so that all lines had identical slopes. The slope gives the dissociation constant of the FMNHg-enzyme com- plex and the intercept the concentration in moles per liter of FMNHz-binding sites.

TABLE I FMMH&nding sites of bacterial lucijerose

Luciferase (figure) Enzyme Concentration of FMNHt-binding concentration0 FlMNHz-binding

sites sites per lucifersse

molecule

MAV (Fig. 5). MAV (Fig. 6) MAV (Fig. 7). MAV (Fig. 5) MAV (Fig. 6) MAV (Fig. 7) Pf (Fig. 9). Pf (Fig. 9).

/ - moles/liter

1.04 X IO-6 1.07 X 10-G 1.04 X 10-O 0.99 X 10-S 1.04 x 10-5 0.90 x 10-G 2.08 x 10-G 1.98 x 10-C 2.08 x lo-” 1.99 x 10-S 2.08 x 10-s 2.00 x 10-6 1.71 X 10-G 1.50 x 10-E 3.42 x 10-6 3.47 x 10-C

1.03 0.95 i 0.116

0.89 f 0.17 0.95 0.96 f 0.06

0.96 f 0.05 0.88 f 0.05 1.00 f 0.02

a The enzyme concentration was based on a specific extinction coefficient of 0.94 (O.lyO, 1 cm) at 280 nm and a molecular weight of 79,000.

b Standard dcviat,ion.

trations well above the Kd for FMNH2 binding. Under these conditions, Equation 2 does not apply since in its development, it was assumed that the free substrate concentration (81) was equal to the total substrate concentration (S). Such an approxi- mation is true only if the concentration of enzyme is much less than the K,j for FMNH2 binding.

Under the conditions of high enzyme concentration, (Sf) is given by

6%) = (S) - VW = 6s) - v/vInar wr) (13)

Substitution in Equation 2 gives the following relationship in three different forms.

V rnSX - = 1 -t [Kd i- f&)(1 - ~/vrn,x)~ l/(s) (14)

V

(S)(~-l)=IC,+(E,)(l-u/v~~~) (15)

0.3

It should be emphasized that (ET) is actually the molar concen- tration of FMNHz-binding sites and not (necessarily) the con- centration of enzyme molecules.

Fig. 5 gives a plot of V,,,/v versus l/S for the experimental data. According to Equation 1.4 at high substrate concentra- tions v approaches Vmax (i.e. (1 - v/Vm,,) -+ 0) and the slope becomes K,J whereas at low substrate concentration v approaches 0 (i.e. (1 - v/V,& -+ 1) and the slope becomes Kd + (ET). Since accurate experimental data can be obtained at low sub- strate concentrations (e.g. at v = 0.003 V,,,), the value of Kd + (ET) can easily be obtained from the limiting slope at high values of l/X in the above plots. At high substrate concentra- tions, the slope decreases and approaches the value of Kd for FMNH2. The Kd value, however, can be more simply deter- mined from the slope at low enzyme concentration ((ET) < Kd). The concentration of FMNHt-binding sites can thus be deter- mined.

Fig. 6 gives a plot of (8) (Vmax/v - 1) versus (1 - v/V,,=) at the three enzyme concentrations (see Equation 15). In this plot the slope gives the concentration of FMNHz-binding sites

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7672 Determination of FMNH=JGading Sites on Bacterial Luciferase Vol. 246, No. 24

FIG. 8. Dependence of the reciprocal of the initial light inten- sity (l/o) on the inverse of the FMNHt concentrations in moles per liter (l/S) for Pf luciferase. The assay mixture contained 2 X 10-81~5 Pf luciferase and the reaction was initiated by the injec- tion of decanal.

directly and the intercept gives the Kd. All three plots intercept the ordinate at the same point. In Fig. 7 the data are plotted according to Equation 16. In this case, the slope is equal to Kd and the intercept on the ordinate gives the concentration of enzyme-binding sites.

A summary of the results obtained in Figs. 5, 6, and 7 is presented in Table I. The data at the two protein concentra- tions analyzed by the three methods agree within experimental error, showing that bacterial luciferase contains only a single binding site for FMNHz per molecule of molecular weight 7.9 x 104.

FMNHz Binding to Pf Lucifererase-It was recently reported that luciferase isolated from a different bacterial strain is dis- tinctly different from MAV luciferase (6, 13). This is referred to as Pf luciferase, and studies were carried out to determine whether its FMNHt-binding characteristics were similar to those of MAV luciferase. This question was of particular interest since the bacterial strain from which Pf luciferase was isolated (P. fischeri, formerly called Achromobacter $fischeri) is presumably similar or identical with that used by McElroy and Green (10). The same assay as described above (dithionite) was used except that the concentration of sonicated aldehyde required for maxi- mum activity was about 10 times greater than that for MAV luciferase. The experimental results are plotted in Fig. 8 and give a linear relationship between l/v and l/S over a wide range of FMNHz concentrations (1 x lOA to 5 x 10eg M). The value obtained for the Kd of Pf luciferase for FMNHz is 9.7 x 1OW M.

I I I 0.2 0.4 0.6 0.0

w-"/v,)

FIG. 9. A plot of S (V,,,/v - 1) versus (1 - v/Vmax) at different Pf luciferase concentrations (2 X 1W M (0); 1.71 X W6 M (0); 3.42 X 10-e M (A)). The experimental data at low enzgme con- centrations (0) d&e fitted with a horizontal line with the mini- mum standard deviation. The data points at high enzyme con- centrations (A and 0) at (1 - v/V,& 5 0.9 were fitted with straight lines intercepting the ordinate at a common point with the minimum standard deviation. The slope gives the concen- tration (M) of FMNHZ-binding sites and the intercept t,he dissocia- tion constant for the FMNHQ-enzyme complex.

Number of FMNH2-binding Sites in Pf Luczyerase-The num- ber of FMNHS-binding sites in Pf luciferase was investigated by enzymatic measurements at high protein concentrations. The experimental data are plotted according to Equation 15 and show a linear relationship between ~(V,,,/V - 1) and (1 - v/V,,,) at light intensities (v) between 10 and 100% of V,,, (Fig. 9). At low substrate concentrations ((8) < 2 x 10e7 M; (1 - v/ V,,,) > 0.9) and high enzyme concentrations (1.7 x low6 and 3.4 X 10e6 M), the points deviate from the linear plot, corre- sponding to measured light intensities that are about 10 and 20% too low for the two respective enzyme concentrations. Although no conclusive explanation is available for these deviations, a con- taminating component which is present at about 2% the molar concentration of Pf luciferase and tightly binds FMNHz would have this effect. At high enzyme and low substrate concentra- tions, this component would effectively lower the substrate con- centration. Such an effect would not be observed either at higher substrate concentrations or low enzyme concentrations. In this regard, it should be noted that a light-induced biolumines- cent activity is present in the Pf luciferase preparation. Al- though the identity and relative amount of this protein compo- nent have not yet been determined, it is not identical with Pf luciferase (5, 14).

The experimental points at higher substrate concentrations ((8) > 2 x 10-T M; (1 - v/V,,,) < 0.9) clearly fall on a linear

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Issue of December 25, 1971 E. A. Meighen and J. W. Hastings 7673

plot. The numbers of FMNHZ-binding sites calculated from the slopes are summarized in Table I. These results show not only that Pf luciferase has only one FMNHZ-binding site, but that any contaminating components must be present in relatively small amounts.

DISCUSSION

Previous investigations on bacterial luciferase indicated that the bioluminescent reaction had a second order dependence on the FMNHz concentration (10). These measurements were con- ducted by the standard assay procedure (injection from a syringe of catalytically reduced FMNH2) over a relatively limited con- centration range (v = 0.5 to 1.0 V,,,). In contrast, the present experiments were conducted by a new assay procedure (dithionite assay: injection of an aldehyde-oxygen suspension into an enzyme solution preincubated with FMNHz and Na2S204) and exhibited a first order dependence on FMNH, concentration over a wide range (0 = 1e3 to 1.0 V,&.

At least two explanations are available for the discrepancy: first, some nonenzymatic oxidation of FMNHz clearly occurs in the syringe prior to injection, and this can be a serious problem. Secondly, the relationship between light intensity and FMNHl concentration is not described by a steady state equilibrium equation in an assay involving the injection of FMNH2. Only if the equilibrium between enzyme and FMNHs is rapid com- pared to the interaction of FMNHz or the FMNHz-enzyme com- plex with oxygen would it have been possible to achieve such a pseudoequilibrium by an FMNHz injection assay. Even under t.hese conditions, the possibility of obtaining a relationship in the form of the Michaelis-Menten equation is restricted to the special case where the nonenzymatic autoxidation rate of FMNHs (k4) is approximately equal to the rate of reaction of oxygen with the FMNHz-enzyme complex (kz). Consequently on both prac- tical and theoretical grounds, the dithionite assay appears to be of greater value for studying the interaction of bacterial luciferase with FMNHz than does the standard injection assay.

In view of the high specificity of bacterial luciferase for reduced flavin mononucleotide (15), the dithionite assay should also pro- vide a convenient and rapid method for its assay in biological material, extending the applicability of a recently reported pro- cedure for detection of FMN in very low amounts (16). Since reproducible enzymatic measurements (v/V,,~) can be made at FMNHa concentrations as low as lOA M, this presently permits as little as 2 x lo-l3 moles of FMN in 2 ml to be detected. The lower limit in the present experiments was entirely due to back- ground activity in the enzyme preparation (about 20% of v at lop9 zvz FMNHs). It should thus be conceivable to extend the assay to much lower FMN concentrations by procedures aimed at removing this background activity.

The determination of flavin-binding sites was carried out at high enzyme concentrations, where it is generally difficult to re- late directly the initial velocity to the substrate concentration. Nevertheless, it is possible to make such a correlation in this case since the ES complex can be formed without initiation of the over-all enzymatic reaction and its conversion to product is more rapid than the binding of additional substrate molecules. Fur- thermore, the nonenzymatic autoxidation of free substrate (FMNHa) by O2 occurs more rapidly than its binding to luciferase. Thus no additional ES complex was formed after the initial con- version of ES to product (ES”). The amount of the stable in- termediate (EXO) formed could then be simply measured by its

subsequent light emission. Consequently it was possible in the present system to correlate directly the initial velocity of the re- action with the substrate concentration at high enzyme concen- trations and hence elucidate the number of substrate-binding sites directly from kinetic measurements.

The experiments clearly show that there is one, not two, bind- ing site per luciferase molecule of 79,000 daltons. This finding is of considerable significance with regard to the mechanism of the bacterial bioluminescent reaction, eliminating mechanisms which require the participation of 2 FMNHz molecules for the emission of a single photon (10, 17). In their proposed mecha- nism, McElroy and Green (10) were especially concerned with the energetic requirements of the reaction, noting that even the oxidation of 2 FMNHz molecules was marginal or inadequate for the emission of a photon at 490 nm. They suggested that the mechanism might also involve the peroxidation of aldehyde to the corresponding acid, thereby providing considerable (~70 kcal) additional energy. Actually, the finding that only a single FMNHz is involved thereby more convincingly implicates alde- hyde oxidation as an integral part of the bioluminescent reaction, since the oxidation of only 1 FMNHz molecule will clearly not provide sufficient energy for photon emission at 490 nm (~60 kcal). Consequently, a mechanism involving the oxidation of aldehyde and a single FMNI-12 molecule would seem likely in the bacterial bioluminescent reaction. However, such a mechanism can only be regarded as tentative until the stoichiometric con- version of aldehyde to acid can be directly related to photon production in the luciferase system. Nevertheless, a model in which aldehyde has only a catalytic role and is not oxidized (18) would not be easily reconciled with the energetic requirements in a single FMNHz mechanism.

Since bacterial luciferase is a heteropolymeric protein contain- ing two nonidentical subunits (6, 19), only one of the subunits is probably intimately involved in the flavin binding. Recent evi- dence indicates that this binding site is specifically located on the a! subunit of luciferase (20, 21). In hybrid luciferase molecules in which only one of the two subunits was chemically modified by succinylation, the Kd for FlMNHt binding was increased in the hybrid with an altered 01 subunit, but unchanged in the hybrid possessing a modified 0 subunit. Furthermore, modification of the OL subunit affected the decay rate of light emission from the stable bioluminescent intermediate, whereas modification of the /3 subunit had no effect. Although this result suggests that the catalytic steps occur on the 01 subunit, further work will be re- quired to determine the specific site of interaction of aldehyde and its role in the bioluminescent reaction.

5.

6.

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Edward A. Meighen and J. Woodland HastingsMONONUCLEOTIDE BINDING TO BACTERIAL LUCIFERASEBinding Site Determination from Kinetic Data: REDUCED FLAVIN

1971, 246:7666-7674.J. Biol. Chem. 

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