ARCHIVES Vol. 185,

OF BIOCHEMISTRY No. 2, January 30,

AND BIOPHYSICS pp. 391-399, 1978

Inactivation of Salmonella by Specific Chemical MARY Department

FEDARKO of Biochemistry,

Phosphoribosylpyrophosphate Modification of a Lysine ROBERTS

AND

of Illinois,

Uniuersit,y Received

ROBERT

August

Urbana,

Synthetase Residue’

L. SWITZER” Illinois

61801

1, 1977

Phosphoribosylpyrophosphate synthet,ase from Salmonella typhimurium contains nine lysine residues per subunit and can be inactivated by reagents specific for this amino acid. Pyridoxal-P reversibly inhibited the enzyme by about 70% by forming a Schiff base derivative with lysine. Reduction with NaBH, made this inactivation irreversible. Kinetic experiments indicated that the failure to inactivate the enzyme completely in a single treatment with pyridoxal-P reflects a reversible equilibrium between inactive Schiff base and a noncovalent complex. Modification of one lysine residue per subunit correlated with apparently total loss of activity. The rate of inactivation of the enzyme was decreased fourfold by saturating concentrations of ATP and was decreased at least 20-fold by formation of a quaternary complex of the enzyme with Mg’+, a,@-methylene ATP, and ribose-5-P. Trinitrobenzenesulfonate also irreversibly inactivated the enzyme, but this reagent was less specific in that the loss of activity corresponded to the modification of four to five lysine residues. These results suggest that an essential lysine is near the active site of phosphoribosylpyrophosphate synthetase.

PRPP4 synthetase (ATP:n-ribose-&phosphate pyrophosphotransferase, EC 2.7.6.1) is a key enzyme in the biosynthesis of purine, pyrimidine, and pyridine nucleotides, histidine, and tryptophan. It catalyzes an uncommon pyrophosphoryl group transfer. Progress has been made in the physical and chemical characterization of the enzyme from Salmonella typhim.urium (1) and in understanding kinetic and mechanistic aspects of the reaction (2-6). I This work was supported by United States Public Health Service Grant AM 13488 from the National Institute of Arthritis, Digestive, and Metabolic Diseases. This is Paper XI in the series “Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase.” 2 Present address: Department of Chemistry (M0161, University of California, San Diego, La Jolla, California 92093. ’ To whom all correspondence should be addressed. 4 Abbreviations used: PRPP, &phospho-o-ribosyla-I-pyrophosphate; TNBS, 2,4,6-trinitrobenzenesulfonate; uv, ultraviolet; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid).

Since both substrates are negatively charged, and Pi is required for catalytic activity and structural stability, it is likely that the enzyme has positively charged residues involved in the binding of these molecules. A basic group could also participate in the catalytic mechanism by assisting in removal of a proton from the anomeric hydroxyl group of ribose-5-P, which would facilitate an attack on the p phosphorus of ATP. The only residue previously shown to be near the active site and essential to enzyme activity is one of four cysteine residues (7). We have used chemical modification to investigate the role of lysine residues in the PRPP synthetase reaction. PyridoxalP has been used as a site-specific reagent to identify reactive lysine residues in enzymes that do not require it as a cofactor for catalysis (8). In this study we have found that pyridoxal-P reacts with one lysine residue per subunit of PRPP synthetase in an active site-directed manner to form an inactive enzyme.

391 0003-9861/78/1852-0391$02.00/o Copyright 0 1978 by Academic All

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392

ROBERTS MATERIALS

AND

AND

METHODS

Enzyme PRPP synthetase was purified from S. typhimurium cells as described previously (1) and had a specific activity of 75-130 ~mol/min/mg of protein in the ““P-transfer assay (3). Protein concentration was determined by the method of Lowry et al. (9), using a color value based on dry weight as described (1). Reagents Pyridoxal-P, pyridoxal, TNBS (Sigma), NaBH, (Alfa Inorganics), and Na-dodecyl-SO, (Fisher Scientific) were used without further purification. ATP, ribose-5-P, and ADP were the best grades available from Sigma. [Y-~‘P]ATP was prepared by the exchange procedure described previously (3). NcY-Acetyl-N-•-phosphorylpyridoxyllysine was prepared by reducing a mixture ofN-a-acetyl-uL-lysine (Sigma) and pyridoxalP with NaBH, and purified by the paper chromatographic system of Raetz and Auld (10). Preparation tase

of Lysine

Derivatives

of PRPP

Synthe-

Pyridoxal-P treatment. Enzyme (0.05-0.30 mg/ml) was incubated with pyridoxal-P in 0.025 M potassium phosphate, pH 7.5, at 25°C for various lengths of time. The time course of inactivation of the enzyme was determined in two ways: (a) assaying aliquots of the incubation mixture with the 32Ptransfer assay for 3-5 min (the short time minimizes dissociation of the covalent Schiff base complex); (b) reducing the Schiff base to a pyridoxamine derivative by treating the sample at 0°C with a drop of octanol and 1 mg of NaBH,/ml. After reduction, the enzyme was dialyzed against 0.05 M potassium phosphate, pH 7.5. Samples of enzyme without pyridoxal-P were incubated and treated in parallel as a control. The enzyme was not affected by treatment with NaBH,; however, prolonged dialysis following the reduction can lead to some activity loss. The quantity of pyridoxamine-P bound to the protein was measured spectrophotometrically at 325 nm, using a molar extinction coefficient of 10,000 for N-6-phosphopyridoxyllysine (11). Trinitrophenylation. Enzyme (0.1-0.3 mg/ml) was incubated with l-25 mM TNBS in 0.025 M potassium phosphate, pH 7.5, at 25°C. The reaction was stopped by the addition of 2-mercaptoethanol or lysine in a 50-fold excess over the TNBS present. Enzyme treated in this way was dialyzed against buffers containing 0.1% Na-dodecyl-SO, to remove free sulfonate and sulfite. A molar extinction coefficient of 14,500 at 345 nm was used to estimate the total number of trinitrophenyl groups of the dialyzed material (12). The reaction could be moni-

SWITZER tored continuously by observing the absorbance a 367 nm [the liberated sulfite changes the absorptior characteristics of the trinitrophenyl group, bu there is an isosbestic point at 367 nm with a molar extinction coefficient of 10,500 (12, 13)]. Aliquots were removed during the reaction for enzyme as says. Absorbance

Measurements

Absorbance measurements were carried out with a Zeiss PMQ II spectrometer or an Acta V spectrometer. Samples were centrifuged at 10,OOOg for 10 min and kept dust free. Unmodified PRPP synthetase shows pronounced light scattering. This is a reflection of the tendency of the enzyme to aggregate, even in dilute solutions (1). Thus, for spectral quantitation of pyridoxal-P or TNBS incorporation into PRPP synthetase the light-scattering contributions from the protein must be determined. To this end, absorbance measurements were extended to 550 nm (where both protein and reagents do not absorb light), and the data were graphed as log A versus log p according to the equation A = kii”, where k is a constant for polymers, fl is the wave number, and n, the exponent, is 4 for particles smaller than 1/20th the wavelength of light (14). The extinction due to scattering was then linearly extrapolated to the uv range and subtracted. Amino

Acid

Analysis

Acid hydrolyses were performed as recommended by Moore and Stein (15). Since the sulthydryl content of enzyme derivatives was checked by titration with 5,5’-dithiobis(2-nitrobenzoic acid) prior to hydrolysis, no special care was taken to preserve cystine in the analysis. Analyses of the hydrolysates were performed using a Beckman Model 120 amino acid analyzer. RESULTS

Reaction of Pyridoxal-P thetase

with

PRPP

Syn-

PRPP synthetase in 0.025 M potassium phosphate, pH 7.5, did not absorb light in the region between 300 and 450 nm. The addition of pyridoxal-P to an enzyme solution resulted in a difference spectrum with maxima at 430 and 270 nm, a minimum at 378 nm, and a shoulder in the 340-nm region, characteristic of pyridoxal-P Schiff bases and aldimines (11, 16). The activity of the enzyme was inhibited by incubation with pyridoxal-P; the residual activity depended on the concentration of pyridoxalP up to 4 mM (Fig. 1). Greater concentra-

LYSINE

INACTIVATION

11Ls-zr4L

OF

PHOSPHORIBOSYLPYROPHOSPHATE

Pyrldcxd F’irnMI

1. The effect of pyridoxal-P concentration on the fraction of initial specific activity. Enzyme (0.06 mg/ml) was incubated for 30 min with the indicated concentrations of pyridoxalP under conditions detailed under Material and Methods. FIG.

tions of reagent produced no greater than 70% inactivation in 30 min of reaction. The inactivation was reversed only slightly (~5%) when the reaction mixture was diluted 50-fold, and an aliquot was added within 15 s to the standard assay mixture and assayed for short periods of time (3-5 min). All activity could be restored by dialysis against phosphate buffer; slow reactivation could be observed upon adding 5 to 10 mM lysine. Formation of a Pyridoxamine-P of PRPP Synthetase

SYNTHETASE

393

lel. After paper chromatography (111, a fluorescent, ninhydrin-positive spot with mobility identical to that of synthetic N-Epyridoxyllysine was observed. Amino acid analysis demonstrated that only lysine was modified (Table I). A preparation of pyridoxamine-P enzyme, labeled with 1.1 pyridoxamine-P per subunit, as judged by the absorbance at 325 nm (prepared with 4 mM pyridoxal-P, incubated for 0.7 h, and reduced with NaBH,), was used for the analysis. The amount of N-e-pyridoxyllysine found on direct analysis was estimated to be 0.9 per subunit after correcting for the small amount of destruction of N-e-pyridoxyllysine seen with the standard; 1.1 mol of N-•-pyridoxyllysine could be deduced from the decrease in lysine residues. Reaction with the N-terminal group is excluded, because the N terminus of PRPP synthetase is a proline residue (11. The cysteine content of the pyridoxal-P-modified enzyme was checked by DTNB titration (7) and found to be unaltered. Varying the concentration of pyridoxalP and time of incubation before NaBH,

Derivative

Correspondence between extent of chemical modification of PRPP synthetase by pyridoxal-P and inactivation was studied by reduction of the Schiff base with NaBH,. The pyridoxamine-P derivative of PRPP synthetase had an absorption maximum at 325 nm (Fig. 21, which was not observed with enzyme that was treated with borohydride alone. To show that lysine residues were modified by pyridoxalP, an enzyme derivative containing 1.2 mol of pyridoxamine-P per mole of subunit (prepared by incubation of the enzyme with 4 mM pyridoxal-P for 1 h, followed by NaBH3 treatment) was hydrolyzed in 6 N HCl. A sample of N-a-acetyl-N-e-phosphopyridoxyllysine was hydrolyzed in paral-

Log

V

(cm’)

FIG. 2. Absorption spectrum of the pyridoxamine-P derivative of PRPP synthetase. Enzyme concentration was 0.4 mg/ml in 0.05 M potassium phosphate, pH 7.5. To quantitate the pyridoxamine-P content, the extinction caused by scattering was extrapolated to the uv range and deducted from the absorbance at 325 nm, as shown by the vertical arrow.

394

ROBERTS TABLE

EFFECT OF PYRIDOXAL-P ACID COMPOSITION

Amino

Lysine Histidine Arginine N-e-pyridoxyllysine

acid

AND

I

REACTION OF PRPP

ON THE AMINO SYNTHETASE

Residues

per subunit

Unmodilied enzyme

Pyridoxamine-P derivative

8.6 3.9 21.4 0

SWITZER

such a change. The reduced derivative was also examined for inhibition by ribose-5-P induced by ADP, which is shown by native enzyme (4). The extent of inhibition paralleled that of native enzyme. Effect of Pyridoxal-P Concentration Znactivation of PRPP Synthetase

7.5 3.9” 21.5 0.9”

on the

A simple reaction scheme for pyridoxalP and enzyme that fits all of our observa-

(1 The N-•-pyridoxyllysine is eluted slightly earlier than histidine and partially overlaps that peak. Assuming that the amount of histidine is not altered in the derivative, one can calculate the N-cpyridoxyllysine content from the difference.

reduction yielded enzyme samples of different activities and pyridoxamine-P content. Figure 3 shows that the loss of up to 70% activity is directly proportional to the incorporation of 0.9 pyridoxamine-P per enzyme subunit. Prolonged incubation periods (3-4 h) modified up to 2.5 lysine residues, but only reduced the activity to 16%. Activity was never reduced to 0% by a single treatment of pyridoxal-P and NaBH,. This indicated either that the modified enzyme had residual activity or that only about 70% of an essential lysine residue was modified. Experiments shown below suggest that the latter is the correct explanation. Kinetic Properties of Pyridoxamine-P rivative of PRPP Synthetase

De-

Table II shows some kinetic parameters of the reduced derivative compared to unmodified enzyme. V was decreased by a factor of 4 after modification; other constants were not significantly altered. Such a result would be expected if a fraction of the enzyme were unmodified and fully active and the rest were modified and completely inactive. The same result would be expected if the kinetic constants for completely modified enzyme changed such that K, was much higher and a range of substrate concentrations too low to detect altered enzyme was used (17). In our case, the range of ATP (0.04 to 1 mM) and of ribose-5-P (0.2 to 2 mM) concentrations should have been sufficient to detect

LL %

3 \ \ 10

d5

Moles Fyr,doxol-P

15

20

lncorporafed/Mole

25

Subuntl

FIG. 3. Stoichiometry of inactivation of PRPP synthetase and incorporation of pyridoxal-I’. The dashed line extrapolates to 1.1 pyridoxamine? per subunit for total loss of activity; the intersection (arrow) indicates that the major loss of activity corresponds to formation of 0.9 pyridoxamineP per subunit. Points beyond 1 pyridoxamine? per subunit were incubated with 5-8 rnM pyridoxal-P for 1.5 to 3 h. TABLE COMPARISON PYRIDOXAMINE?

Kinetic

II

OF KINETIC

PROPERTIES

DERIVATIVE WITH PRPP SYNTHETASE

constant

V (fimol/min/mg) K,, ATP (mM) K,, ribose-5-P (rnM) Ki, ATP (rnM)

a Unmodified zyme 85.2 0.037 0.13 0.09

2 k f -t

enb 5.1 0.008 0.05 0.06

OF THE UNMODIFIED

Pyridoxamine-P derivative 20.0 0.034 0.15 0.12

t + ? _t

’ 1.8 0.010 0.06 0.09

(I The values and their standard errors were obtained from computer tits of the data with the SEQUEN program as previously described (4). Substrate concentrations were: ATP, 0.04 to 1 mM; ribose-5-P, 0.2 to 2 mM. h The unmodified enzyme used as a control was incubated at 25°C without pyridoxal-P, reduced with NaBH,, and dialyzed exactly as the pyridoxal-Ptreated sample. r The enzyme contained 1.1 mol of pyridoxamineP per subunit as judged by absorbance at 325 nm.

LYSINE

INACTIVATION

tions is that suggested (18)“: k E + P\eE...P. 1

OF

PHOSPHORIBOSYLPYROPHOSPHATE

395

SYNTHETASE

by Chen and Engel k,, I k-. E = P, 2

where E. . . P is a noncovalent complex of enzyme and pyridoxal-P and E = P is the covalent Schiff base. Upon dilution into the assay, the noncovalent complex is assumed to dissociate immediately and is measured as active enzyme, while the Schiff base is assumed to be inactive. If the formation of the noncovalent complex is kinetically significant, k,,,,, the apparent first-order rate constant, will show saturation behavior as a function of pyridoxal-P concentration. Such behavior is evidence for a discrete binding site for pyridoxal-P on the enzyme. A double-reciprocal plot of k,,, versus pyridoxal-P concentration gives a straight line, where l/k+, and k+,/kp, are the intercepts of the ordinate and abscissa, respectively. Figure 4 shows the saturation behavior of k,,,,, as a function of pyridoxal-P concentration for PRPP synthetase. The rate of formation of Schiff base, k,,, and the dissociation constant for pyridoxal-P, K (== k~,lk+J, are calculated to be 0.12 min..’ and 0.79 mM, respectively. In this model two kinds of kinetic behavior can be distinguished depending on the stability of the pyridoxal-P-enzyme Schiff base. For enzymes that react with pyridoxal-P, if kps, the rate of breakdown of the Schiff base, is much less than k+2, the rate of formation, inactivation by any concentration of pyridoxal-P will produce the same residual activity. On the other hand, if k-~, is not much less than k+2, the minimum attainable activity in a single reaction cycle with pyridoxal-P and subsequent reduction with NaBH, is determined by the fraction (k_,)l(k+, + k-,), and the residual activity will depend on the pyridoxal-P concentration at subsatur5 Other, more complex schemes involving formation of forms of the pyridoxal-P-enzyme complex that react slowly with NaBH, can also be imagined, but in the absence of experimental evidence for the presence of such forms we have chosen the simplest scheme.

,.;i

2 -y, ,0x,, ~0 rnw /

6

h

FIG. 4. The effect of pyridoxalP concentration on the pseudo-first-order rate constants of enzyme inactivation. Enzyme (0.06 mgimll was incubated with the indicated concentrations of pyridoxalP and an aliquot was assayed at various times as described under Materials and Methods. The initial slope of the log (enzyme activity) versus time was used to derive k,,,,, at each concentration. For pyridoxal-P concentrations greater than 2 mM, only the first 4 min of data were used (longer times showed nonlinearity as the equilibrium between noncovalent complexes and the covalent Schiff base was established).

ating values. Reversal of the pyridoxal-pPRPP synthetase complex (without addition of NaBH,) could be demonstrated directly from reactivation experiments (Fig. 51, which permit an estimate for k. p of 0.05 min-’ (18). In this experiment, dilution of the enzyme 50-fold should shift the position of the equilibrium far in favor of unbound pyridoxal-P. The values of kmz and k,, predict that the minimum attainable activity is 0.33 of the initial activity, a value which agrees well with experimental results for incubation times under 1 h and high concentrations of pyridoxal-P. As further evidence that this equilibrium determines the residual activity of PRPP synthetase, we have inactivated the enzyme by repetitive treatments of pyridoxal-P and NaBH,. Table III shows the results of four successive incubations with 4 mM pyridoxal-P for 1 h. Each incubation decreased the activity to 0.36 of the previous value; the final activity was 0.03 of the initial value. Taken together, the data suggest that reaction of a single lysine residue of PRPP synthetase with pyridoxal-P causes complete or nearly complete inactivation of the enzyme. As the data in Fig. 3 show, other lysine residues can also react with

396

ROBERTS

AND

FIG. Time (mini

5. Reactivation of PRPP synthetase by dilution after inactivation with pyridoxalP. Enzyme (0.3 mglml) was incubated with 4 rnM pyridoxal-P. After 25 min, 0.10 ml of this mixture was diluted 50-fold into 0.025 M potassium phosphate buffer, pH 7.5. Three-minute assays were conducted at the indicated time intervals to determine enzyme activity. TABLE REPETITIVE

TREATMENTS

DERIVATIVE

Number treatments

of

III OF THE

PYRIDOXAMINE-P

WITH 4 mM PYRIDOXAL-P REDUCTION BY NaBH,”

Fraction specific

of initial activity

AND

Predicted fraction based on equilibrium at first treatment b

0 1

1.00

0.36

0.36

2 3 4

0.11

0.13

0.06 0.03

0.05 0.02

1.00

n Enzyme (0.4 mg/ml) was made 4 mM with pyridoxal-P, incubated for 1 h at 25”C, and then reduced with NaBH, and dialyzed for about 4 h to remove small molecules This was repeated four times. A control without pyridoxalP was treated in parallel to account for the gradual loss of activity the enzyme undergoes upon prolonged dialysis. b Calculated from the assumption that each treatment reduces the activity to 0.36 of the value previously obtained.

pyridoxal-P, but they react only at high concentrations of pyridoxal-P and with a long reaction time; modification of these residues has relatively little effect on enzyme activity. Effects of Substrates and Pi on the Rate of Inactivation of PRPP Synthetase by Pyridoxal-P Table IV shows the apparent rate constants for inactivation of the enzyme in the presence of various substrates. The

SWITZER

inactivation was inhibited when 5 mf Mg2+ was included, although this could be caused by Mg’+ complexing pyridoxal-P and altering the reagent’s reactivity toward lysine. Addition of Mg-ATP further decreased the rate to one-fourth of the initial value. The most pronounced effect was seen with the Mg’+, o+/3-methylene ATP, and ribose-5-P-enzyme complex; almost no inactivation occurred in 30 min. These results suggest that the essential lysine residue is protected from reaction when the active site is occupied. The observation that ATP alone provided only moderate protection suggests that the lysine residue in question may be near the ribose-5-P site. When ribose-5-P and Mg’+ were added, no effect greater than that with Mg’+ alone was observed. Increasing concentrations of Pi also exerted a moderate protective effect; k,,, was found to be inversely proportional to Pi concentration in the range of from 5 to 100 mM (data not shown). The apparent K,, for Pi was calculated to be 18 mM. Reactivity of Trinitrobenzenesulfonate with PRPP Synthetase PRPP synthetase was quite susceptible to inhibition by TNBS. The addition of four to five trinitrophenyl groups per subunit led to total loss of activity (Fig. 6). In contrast to pyridoxal-P, inactivation by TABLE EFFECT

OF LIGANDS PYRIDOXAGP

Ligand None

5 mM MgCl* 5 mM MgCl,, 5 mM MgCl,,

IV

ON THE RATE OF REACTION WITH PRPP SYNTHETASE’

2 mM ATP 2 mM cY,fi-methylene ATP, 5 mM ribose-5-P 5 mM MgC&, 5 mM ribose-5-P

k appb 2 SE (min

OF

‘1

0.058 t 0.004 0.023 t 0.002 0.014 f 0.002 0.001

t 0.002

0.023 k 0.004

o Enzyme (0.06 mglml in 25 mM phosphate buffer, pH 7.5, 25°C) was reacted with 1 mM pyridoxalP in the presence of ligands as shown. Aliquots for assay were removed to follow the reaction time course. b The reaction is pseudo first order, but begins to deviate from this as the reaction progresses and the reverse reaction (k_,) becomes more significant. Therefore, k,,,,, has been determined from the early time _ noints.

LYSINE

INACTIVATION

OF

PHOSPHORIBOSYLPYROPHOSPHATE

397

SYNTHETASE

enzyme under nondenaturing conditions showed that the lysine residues modified by pyridoxal-P are a subset of the ones reactive with TNBS. TNBS was not as selective as pyridoxal-P and modified about half of the total lysine residues when it reacted with the native enzyme. DISCUSSION Moles

TNES

FIG. 6. Stoichiometry synthetase by TNBS. scribed under Materials

lncorporoted/Mole

Enzyme

of inactivation Reaction conditions and Methods.

of PRPP are de-

TNBS was first order in reagent concentration (0.5 to 25 mM); the bimolecular rate constant calculated from the data was 17 M-’ min’ in 25 mrvr phosphate buffer, pH 7.5, at 25°C. Thus, an intermediate noncovalent complex of the enzyme with TNBS was not kinetically significant in the reagent concentration range studied. The inactivation rate of TNBS was affected by substrates in the following manner: 5 mM MgCl, had no effect, 2 mM MgATP decreased the rate 14.5-fold, and the quaternary complex with 5 mM MgCL, 2 mM (Y,fi-methylene ATP, and 5 mM ribose5-P had no effect beyond that of Mg-ATP alone. TNBS can react with thiols as well as amino groups, but the reaction is reversible by the addition of 2-mercaptoethanol or by dialysis. The inactivation of PRPP synthetase by TNBS was not reversed by either of these methods. Therefore, under the reaction conditions used, inactivation is probably caused by modification of one or more lysine residues. The total lysine content of PRPP synthetase was measured by trinitrophenylation of the enzyme denatured in 2% Na-dodecyl-SO,. This method gave 8.8 lysine residues per subunit, which is in agreement with the amino acid analysis values of 9 (this study) and 10 (1). Enzyme reacted with pyridoxal-P and reduced with NaBH, can also be treated with TNBS under denaturing conditions to measure the remaining lysine content (Table V). Trinitrophenylation of unmodified enzyme and the two different preparations of pyridoxamine-P

Reaction of one of the 9 or 10 lysine residues of the PRPP synthetase subunit with pyridoxal-P causes a pronounced loss in catalytic activity. Under more forcing reaction conditions additional lysine residues also react with pyridoxal-P, but with little effect on the remaining catalytic activity of the enzyme (Fig. 3). We have tentatively concluded that modification of the first lysine residue is responsible for complete inactivation of PRPP synthetase TABLE QUANTITATION

Enzyme

V

OF LYSINE CONTENT SYNTHETASE WITH TNBS

form

Denatured’ Unmodified enzyme Pyridoxamine-P enzyme (preparation 1) Pyridoxamine-P enzyme (preparation 2) Native Unmodified enzyme PyridoxamineP enzyme (preparation 1) Pyridoxamine-P enzyme (preparation

TNP-lysine’;

IN PRPP

PyridoxamineP-lysine h

Total lysine reacted

8.8” 8.3

0.3

8.8 8.6

6.7

2.3

9.0

5.0 4.2

0.3

5.0 4.5

2.4

2.3

4.7

2) u The TNP-lysine content was measured by absorbance at 367 nm (TNP, trinitrophenyl). b Incorporation of pyridoxalP was determined from the absorption at 325 nm, before treatment with TNBS. ’ Enzyme was denatured in 2% Na-dodecyl-SO, at 37°C for 4 h after treatment with pyridoxal-P and NaBH,, but before reaction with TNBS. d Moles per subunit.

398

ROBERTS

AND

and that the remaining activity is due to the presence of enzyme in which this lysine residue is not modified. This conclusion is based on two lines of evidence. First, the kinetic constants of the modified and native enzyme, determined over a wide range of substrate concentrations, were the same within experimental error, except for the apparent maximal velocity. This evidence is not conclusive because the complete set of kinetic constants was not determined. Second, the kinetics of the reaction of pyridoxal-P with PRPP synthetase and reactivation of the enzyme on dilution were consistent with a simple model in which the residual activity is determined by the equilibrium between the noncovalent pyridoxal-P-enzyme complex and the pyridoxal-P-enzyme Schiff base. The inability to inactivate the enzyme with a single treatment of pyridoxalP and NaBH, is viewed as a consequence of this equilibrium. The fraction of activity surviving successive treatments with pyridoxal-P is in excellent agreement with the fraction calculated from independent determination of the rate constants that determine this equilibrium. It is unlikely that stepwise loss of activity of successive treatments with pyridoxal-P is due to modification of other lysines, because we have shown that such modification does not inactivate the surviving activity in single treatment experiments (Fig. 3). If this analysis is correct, a single lysine residue of PRPP synthetase appears to be essential for activity. The results of substrate protection studies suggest that the lysine residue modified by pyridoxal-P lies at or near the active site of the enzyme, although protective effects caused by a substrate-induced conformational change cannot be excluded. The quaternary complex analog was much more effective than Mg-ATP alone, providing almost total protection against inactivation. This further suggests that the lysine residue lies near a ribose5-P subsite. Modification of lysine by pyridoxal-P involves the introduction of a bulky, anionic group, and less drastic modification might allow activity to be retained. Be-

SWITZER

cause of this one cannot conclude that lysine participates directly in substrate binding or catalysis. Pyridoxal-P exhibits partial selectivity for the “essential” lysine residue, when compared to another anionic reagent such as TNBS. The latter reagent inactivates enzyme without the formation of a kinetically detectable noncovalent complex, and it is less specific in that four lysines are modified at an equivalent rate. Thus, the selectivity seen with pyridoxal-P could reflect either a degree of active site directedness because of the presence of the phosphate group or a specific reactivity of the lysine [i.e., an unusually low pK,,, as has been shown by Piszkwiewics and Smith (19) for glutamate dehydrogenasel. ACKNOWLEDGMENT We Illinois,

thank Dr. Thomas Baldwin, for running the amino acid

University analysis.

of

REFERENCES 1. SCHUBERT, K. R., SWITZER, R. L., AND SHELTON, E. (1975) J. Biol. Chem. 250, 7492-7500. 2. SWITZER, R. L. (1970) J. Biol. Chem. 245, 483495. 3. SWITZER, R. L. (1971)J. Bid. Chem. 246, 24472458. 4. SWITZER, R. L., AND SOGIN, D. C. (1973)J. Biol. Chem. 248,1063-1073. 5. SWITZER, R. L., AND SIMCOX, P. D. (1974) J. Biol. Chem. 249, 5304-5307. 6. MILLER, G. A., JR., ROSENZWEIG, S., AND SWITZER, R. L. (1975) Arch. Biochem. Biophys. 171, 732-736. 7. ROBERTS, M. F., SWITZER, R. L., AND SCHUBERT, K. R. (1975) J. Biol. Chem. 250, 5364-5369. 8. MEANS, G. E., AND FEENEY, R. E. (1971) Chemical Modification of Proteins, pp. 132-135, Holden-Day, San Francisco. 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. RAETZ, C. R. H., AND AULD, D. S. (1972) Biochemistry 11, 2229-2236. 11. FORREY, A. W., OLSGAARD, R. B., NOLAN, C., AND FISCHER, E. H. (1971) Biochimie 53, 269281. 12. GOLDFARB, A. R. (1966) Biochemistry 5, 25702574. 13. PLAPP, B. V., MOORE, S., AND STEIN, W. H. (1971) J. Biol. Chem. 246, 939-945. 14. DOTY, P., AND GEIDUSCHEK, E. P. (1953) in The

LYSINE

INACTIVATION

Proteins (Neurath, p. 406, Academic

OF

PHOSPHORIBOSYLPYROPHOSPHATE

H., and Bailey, V., eds.), Press, New York. 15. MOORE, S., AND STEIN, W. H. (1963) irk Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 819-831, Academic Press, New York. 16. METZLER, D. E. (1957) J. Amer. Chew. Sot. 79,

SYNTHETASE

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Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by specific chemical modification of a lysine residue.

ARCHIVES Vol. 185, OF BIOCHEMISTRY No. 2, January 30, AND BIOPHYSICS pp. 391-399, 1978 Inactivation of Salmonella by Specific Chemical MARY Departm...
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