Kinetic Investigations on a Flavoprotein Oxygenase, 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase*

In the presence of NADH and 0 2 , 2-methyl-3-hy-droxypyridine-5-carboxylate oxygenase (EC 1.14.12.4) from Pseudomom sp. MA-1 catalyzes reductive oxygenation of 2-methyl-3-hydroxypyridine-5-carboxylate (Cpd I) to yield a-N-(acetylaminomethy1ene)succinic acid (Cpd A). Steady state kinetic data and studies with alternate substrates are consistent only with an or- dered mechanism in which Cpd I binds first, followed by NADH; the first product, NAD’, is then released. This event is followed by oxygen binding, and finally release of the oxygenated and reduced cleavage prod- uct, Cpd A. This kinetic mechanism was confirmed by studying inhibition by NAD’, which binds competi- tively with oxygen, but not with NADH. The kinetic mechanism of this reaction resembles that proposed for bacterial flavin monooxygenases that catalyze hydroxylation of aromatic homocyclic compounds. catalyzes the reaction shown in Equation 1 (I). This oxygenase

' These studies were supported by grants from the Robert A.
Welch Foundation (Grant F-714) and the United States Public Health Service (Grants AM 19898 and A I 13940). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Materials-Pseudomonas sp. MA-1 (ATCC No. 33286) was grown on a synthetic medium supplemented with 0.2% pyridoxine (2). Cpd I oxygenase was purified from cells harvested in the late log phase.
The procedure of Sparrow et al. (1) was followed except that 10 mM dithiothreitol was used in place of 2-mercaptoethanol throughout the purification scheme. Cpd I and 5-pyridoxic acid were isolated and purified from culture fdtrates of Pseudomonas sp. MA-1 and Pseudomonas sp. IA, respectively, by procedures described elsewhere (3,4). NADH and NAD+ (Boehringer) and NMNH (Sigma) were from commercial sources.
Assay-Cpd I oxygenase was assayed either spectrophotometrically by absorbance measurements at 340 nm or by use of a Gilson oxygraph equipped with a Clark electrode (ox 15259). For spectrophotometric assays, the standard reaction mixture contained 0.2 mM NADH, 40 PM FAD, and approximately 10 pg of enzyme in a total volume of 1.0 ml of 0.05 M potassium phosphate buffer, pH 8.0. After 2 min at 25 "C, the reaction was initiated by adding 0.2 pmol of Cpd I. Absorbance changes at 340 nm result from disappearance of both Cpd I and NADH: this complication could be circumvented either by dual wavelength measurements (5) or by assays in the oxygraph. For NADH and 0.5 mM Cpd I in a total volume of 2 ml of the buffer; oxygraph assays, the standard reaction mixture contained 0.5 mM other additions, pH, and temperature were identical with those described for spectrophotometric assay.
Data Processing-Double reciprocal plots of initial velocities uersus substrate concentrations were analyzed by Cleland's method (6) using a BASIC Program written for this purpose by Dr. W. L. Lopatin.
Eadie plots with alternate substrates were drawn by eye. The nomenclature used in this paper is that of Cleland (7,8).

RESULTS
Lineweaver-Burk plots in which concentrations of Cpd I and NADH are varied and that of 0 2 is fixed are shown in Fig. 1. The lines converge to a common point on the horizontal coordinate at -20.2 m"', when Cpd I is the variable substrate and NADH is the fixed variable substrate (Fig. IA). When NADH is the variable substrate and Cpd I is the fixed variable substrate, the plots intersect at a common point above the xcoordinate at a value of -9.6 mM" (Fig. 1B). Such plots are indicative of a sequential addition of the two substrates (7, $1, in this case Cpd I and NADH. The intercept replot of each of these plots is shown as an inset in the respective figures. Such plots will be referred to as secondary plots and Lineweaver-Burk plots as primary plots. Primary plots in which the concentrations of O2 and Cpd I were varied with NADH fixed ( Fig. 2A) and the concentrations of NADH and O2 were varied at a fixed concentration of Cpd I (Fig. 2B) are also shown. Both plots yielded a series of parallel lines, consistent with a mechanism involving two partial reactions. The secondary plots are shown as insets in the respective figures.
Of several possibilities, mechanism I, designated Bi Uni Uni Uni Ping Pong by Cleland (7,8), fits these plots best, where C is O?, A and B are reactants yet to be assigned, and P and Q are products. Two substrates, NADH and Cpd I, interact sequentially with the enzyme to generate a complex which fist releases P and then interacts with the third substrate, 0 2 , in a distinct partial reaction.
The initial rate equations, 2-5 (see Supplement) ,' for Mechanism I were derived by the King-Altman procedure (9) for the interconversion pattern shown in Fig. 3. To determine the values of the kinetic constants, it is necessary to identify A and B. This was done as follows: ( a ) The rate of oxidation of NADH (and reduction of enzyme-bound FAD) in the absence of Cpd I is negligible (<0.5%) compared to that observed in the presence of Cpd I (1). It is likely, therefore, that NADH is not the first substrate to interact with the enzyme. However, the possibility that NADH is bound in the absence of Cpd I but cannot reduce the flavin efficiently under these conditions is not excluded by this observation. that Cpd I can interact with the free enzyme in the absence of both NADH and 02. NADH does not perturb the spectrum of the oxygenase in the absence of Cpd I, but this does not rule out binding of NADH by the free enzyme. ( c ) The order of binding of substrates can be determined kinetically by the use of alternate substrates (1 1-13). Such experiments, described below, proved that Cpd I was the fist substrate to interact with the oxidized enzyme under steady state conditions.

5-Pyridoxic Acid and Cpd
I-In the presence of 0 2 , 5pyridoxic acid stimulated NADH oxidation by the oxygenase in a manner similar to Cpd I (1). Little or no hydrogen peroxide (assayed by the method of White-Stevens and Kamin (14)) appeared in such reaction mixtures and since the spectral changes observed in the presence of a NADH regenerating system showed a steady decrease in absorption a t 323 nm and an increase at 260 nm similar to those observed with Cpd I as a substrate, we concluded that 5-pyridoxic acid was a true (but sluggish) analogue substrate for the oxygenase.
Cpd I and 5-pyridoxic acid are closely similar compounds, as are also their reaction products, and we could not devise any sensitive procedures to follow the rate of the oxygenase reaction with one substrate in the presence of the other. We therefore measured the combined rate with the two substrates and derived the rate equation 6 for the case where A is the variable substrate on the assumption that Cpd I ( A , Fig. 4  Oxygen concentration in the oxygraph reaction vessel was varied by mixing appropriate amounts of buffers saturated with nitrogen or air to yield a final volume of 2 ml. The concentrations of enzyme and FAD were the same as those listed in Fig. 1. The concentration of NADH in A and Cpd I in B was 1 mM and 0.5 mM, respectively. Concentrations indicated above the lines correspond to those of Cpd I in Fig. 2A and of NADH in Fig. 2B concentrations of 5-pyridoxic acid and fixed concentrations of NADH and 0 2 are clearly nonlinear (Fig. 5 A ) .
Equation 6 can be linearized to yield equation 7. In accordance with predictions of equation 7,

plots of Au versus A u / [ A ]
are linear (Fig. 5B) and thus justify the assumptions made in deriving equations 6 (Fig. 6A) conform to these patterns. When O2 is the variable substrate, C, the rate equation in the presence of 5-pyridoxic acid has the form shown in equation 9. Plots of the data at several oxygen concentrations (Fig.  6B) are linear as predicted by equation 9 suggesting the basic validity of the assumptions made in deriving these equations.
NMNH As a Substitute for NADH-NMNH effectively replaces NADH as a substrate for Cpd I oxygenase (equation 1) and, since there was no significant uncoupling of its oxidation from Cpd I oxygenation, the mechanism of the oxygenase reaction was assumed to be identical with either NADH or NMNH as substrates (see Fig. 7 ) . If Cpd I is the first substrate to interact with the oxidized enzyme, and is also the variable substrate, rate equation 10 is obtained. The experimental data for the case where NADH, NMNH, or both are present all give linear plots (Fig. 8A), as predicted by this equation, thus confirming that during steady state turnover Cpd I interacts with the free enzyme.
If NADH is the variable substrate, B, and NADH and NMNH are assumed to interact only with the E-Cpd I complex and not with the free enzyme, equation 11 is obtained. This equation predicts that plots of u uersus v/[NADH] wiIl be nonlinear when NMNH is present, in accord with the experimental data (Fig. 8B, curues 1 and 2). Equation 11 is similar to equation 6 and can be linearized in the same way.

The linearized form predicts that a plot of Au versus A u / [ B ] ,
where Au = ub/d and b / d is the observed velocity when only NMNH is present, should be linear, again in accord with experimental observation (Fig. 8B, curues 3 and 4 ) . If NADH were bound by both free and Cpd I-bound enzyme, the rate equations (not shown) would be nonlinear even after transformation.
With O2 as a variable substrate, C, the rate equation takes the linear form shown in equation 12. Eadie plots of velocity uersus oxygen concentration (Fig. 9) are linear and nearly parallel to each other, emphasizing the basic correctness of the general kinetic Mechanism I and the more specific Mechanism 11 for the action of Cpd I oxygenase.
No evidence has yet been presented to show that NAD' release occurs prior to 0 2 binding. Since for p-hydroxybenzoate hydroxylase from P . fluorescens, NADP' release is reportedly independent of 0 2 binding (16), this point was clarified by studies of product inhibition.

Product Znhibition of Cpd Z Oxygenase
At concentrations up to 10 mM, Cpd A did not inhibit the oxygenase reaction, whereas added NAD+ (>I mM) did inhibit. Mechanism I1 predicts that if NAD' release precedes and is essential for Oz binding, NAD' and O2 should interact competitively with the enzyme. Data of Fig. 10 show this to be the case. Variations in concentrations of Cpd I and NADH did not affect the nature of the inhibition by NAD' with respect to 02. This too is in accord with rate equation 13 which relates u to [NAD'] and [OZ]. NAD' affects only the term that includes Kc, and hence should act as a competitive inhibitor with respect to 0 2 . NAD' is a noncompetitive inhibitor with respect to NADH at all concentrations of Cpd I and unsaturating concentration of O2 (Fig. 11A). This finding also supports mechanism 11, for which rate equation 14 relates NAD' inhibition to [NADH]. Replots of intercepts and slopes of curves in Fig. 11A against [NAD'] should be and are linear (Figs. 11B and 11C). The negative x-intercept of these plots is related to kinetic constants as shown in equations 15 and 16. From these relationships, the ratios of rate constants, k-2/ kz and k3/k-3 can be obtained, provided the values of the kinetic constants, K,, K b , and Kc are known. These constants were determined as described below.

Kinetic Parameters of the Oxygenase Reaction
Rate equations 3,4, and 5 show that both the intercept and slope of the secondary plots of Figs. 1 and 2 contain terms that include K,,, and V. For this reason, it is not possible to evaluate these kinetic constants accurately from such plots alone. To circumvent this problem, experiments were performed in which Cpd I was the variable substrate, and NADH and O2 were "fixed" variable substrates added at concentrations such that [NADH]/[02] = 1. Under these conditions,    (Table I) can be calculated. Rate constants kl and k-1 can be calculated by equations 19 and 20, respectively. Ratios k-,/k, and k3/k-3 can be calculated from NAD' inhibition data (equations 15 and 16). These values are presented in Table 11.

DISCUSSION
All of the data presented in this paper support Mechanism I1 for the turnover of the dioxygenase. This mechanism involves two different ternary complexes (E,. -Cpd I .NADH and E r e d m Cpd I -0 2 ) for the Ter Bi reaction catalyzed by the oxygenase. Closely related mechanisms have been proposed for several flavoprotein monooxygenases (17)(18)(19)(20).
The fact that Cpd I participates in both ternary complexes may be important from the physiological standpoint of the organism. If NADH were to reduce the enzyme in the absence of Cpd I, it could lead to the formation of H202, since reduced enzyme reacts with 0 2 in the absence of Cpd I to form H202. The extremely slow rate of reduction of the FAD-enzyme by NADH in the absence of Cpd I ensures that such wasteful pathways do not operate. The rate of reoxidation of FADH2enzyme is also stimulated by Cpd I (1). Cpd I thus has the capacity to interact with both oxidized and reduced forms of the enzyme and efficiently channel electrons toward oxygen activation for the oxygenation reaction.
In interpreting the data with alternate substrates, only the linear or nonlinear nature of the plots was used as evidence for mechanism 11. Cooperativity in binding of any of the substrates could not be detected in initial velocity studies with either physiological or alternate substrates. A random mechanism for binding of Cpd I and NADH was excluded, since such a mechanism would yield nonlinear plots in both Fig. 5B and Fig. 8.
Since NADH does not form a catalytically active complex with free oxygenase during steady state turnover, binding of Cpd I must increase both the affinity for NADH and its reactivity in the ternary complex. The anaerobic rate of reduction of E-FAD by NADH in the presence of 5-pyridoxic acid is only 3.8% of that observed with Cpd I (l), and this low rate probably explains the low rate of the overall reaction with the former substrate. Reoxidation of the reduced enzyme by oxygen is extremely rapid in the presence of either sub-~t r a t e .~ Few studies of oxygenase inhibition by products have been described. Inhibition by protocatechuate was used as an aid in elucidating the mechanism of action of p-hydroxybenzoate hydroxylase (16). NAD' did not inhibit the latter enzyme, a result interpreted to mean that addition of 0 2 to the reduced enzyme is independent of NAD' release (16). However, if (as in the case studied here) inhibition by NAD' were competitive with 0 2 , inhibition by NAD' might not be observed at saturating concentrations of 0 2 . NADP' is a product inhibitor of mammalian microsomal amine oxidase (23), but the kinetic G. M. Kishore and E. E. Snell, unpublished observations. mechanism of this reaction is ordered Ter Bi with NADPH being the f i s t substrate to complex with the free enzyme and NADP' the last product to leave the active site. In contrast to bacterial enzymes so far described, the flavin-oxygen adduct is formed prior to the binding of the amine substrate.
Many kinetic investigations on multisubstrate reactions have assumed that K,,, and V values can be obtained from the x-and y-intercepts of secondary plots when the concentrations of only 2 substrates are varied (e.g. 14). This is strictly true only if fiied substrates are not inhibitory and are present at sufficiently high concentrations so that K , / [ S ] is negligible. We have avoided this problem by measuring initial velocities at constant ratios of the fixed substrates. Under these conditions, secondary plots extrapolate to the true value for 1/V. For data reported in Fig. 12, V is 0.1493 mM/min, which corresponds to a molecular activity of 2407/min at 25 "C and pH 8.0. This value is in the same range as those reported for several flavoprotein monooxygenases (19,21,22).
These studies show that Cpd I oxygenase acts by a kinetic mechanism similar to that by which several bacterial flavooxygenases catalyze hydroxylation reactions, i.e. initial binding of the aromatic substrate followed by NADH oxidation and the reaction of reduced flavoenzyme with 0 2 , presumably to generate a reduced flavin-oxygen adduct which participates in oxygenation of the bound aromatic substrate (24). Whether the reductive ring-cleavage reaction described here also proceeds through intermediate formation of a dihydroflavin peroxide is not yet known.
In the two instances so far examined, the pyridine ring is cleaved by flavoproteins (l), whereas the benzene nucleus in all of a much larger number of instances is invariably cleaved by iron-containing dioxygenases (25). The basic reasons underlying this difference in the cofactor requirement of what appear superficially to be similar reactions remain to be established.