myo-Inositol-1-phosphate synthase from pine pollen: Sulfhydryl involvement at the active site

OF BIOCHEMISTRY AND BIOPHYSICS Vol. 231, No. 2, June, pp. 372-377, 1984

ARCHIVES

myo-lnositol-1 -phosphate Synthase from Pine Pollen: Sulfhydryl Involvement at the Active Site’ SUBHASH Institute

C. GUMBER,

MARY

W. LOEWUS,

AND

FRANK

A, LOEWUS’

of Biological Chemistry and the Program in Biochemistry Washing&m State University, Received November

Pullman,

and Biophysics, Washing&m 99164-63~0

4, 1983, and in revised form January

27, 1984

mgo-Inositol-l-phosphate synthase [EC 5.5.1.4; lL-myo-inositol-l-phosphate lyase, (isomerizing)] from Pinus pomkrosa pollen has been partially purified and characterized. It has a pH optimum between 7.25 and 7.75. The k, for D-glucose 6-phosphate (NAD+ constant at 1 mm) is 0.33 mM. Inhibition by p-chloromercuribenzoate and N-ethylmaleimide, and partial protection against this inhibition by D-glucose 6-phosphate in the presence of NAD+, suggests that there is sulfhydryl group involvement at the substrate binding site. my/o-Inositol-l-phosphate 5.5.1.4; 1~ - myo - inositol

synthase [EC - 1 - phosphate

I

lyase(isomerizing)] catalyzes the conversion of glc-6-P3 to MI-l-P by the partial reactions shown in the following scheme:

6H

OH 1

The enzyme has been purified to homogeneity from plant, animal, and fungal sources (l-6), and many of its properties are now described (7). Virtually all research on the plant synthase has involved the use of angiosperms, although it is well recognized that gymnosperms also produce certain cyclitols, notably sequoyitol, Dpinitol, and MI, in substantial amounts (810). In the present study, pollen from Pinus punderosa was selected as a source for isolation and partial characterization of syn-

Chemicals. PCMB, NEM, and 2-mercaptoethanol were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin; MI, @-NAD+, and glc-6-P from Sigma Chemical Company, St. Louis, Missouri; and quebrachitol, D-chiro-inositol, and L-chit-o-inositol

r Scientific Paper No. 6547, Project 0266, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164. This investigation was supported in part by the National Institutes of Health Grant GM-22427. * To whom correspondence should be addressed.

a Abbreviations used: synthase, mgo-inositol-lphosphate synthase (EC 5.5.1.4); glc-6-P. D-glUCOSe 6phosphate; MI, myo-inositol; MI-l-P, lL-my@inositoll-phosphate; PCMB, p-chloromercuribenzoate; NEM, N-ethylmaleimide; DTT, dithiothreitol.

0003-9861/84 $3.00 Copyright All rights

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

thase. In the course of this study new evidence was obtained that suggests an involvement of sulfhydryl at the substrate binding site of the enzyme. MATERIALS

372

AND

METHODS

SULFHYDRYL

INVOLVEMENT

373

IN myo-INOSITOL-l-P-SYNTHASE

from Calbiocbem, San Diego, California. Z-O,C-Methylene-MI, myo-inosose-2, scylbinositol, and deoxyse&o-inositol ‘were prepared in this laboratory. DPinitol was a gift from E. A. McComb, University of California, Davis. [l-i4C]Glc-6-P was purchased from New England Nuclear Corporation, Boston, Massachusetts. Polk Dehiscing strobiles from P. pono!erosu Dougl. ex P. Laws and C. Laws (western yellow pine) were dried on stainless-steel screens. Released pollen was passed through a No. 20 sieve and stored at -20°C in closed plastic vials (20 g/container). A single strobile produced arbout 9% of its fresh weight as pollen. Enzyme assay. A radioisotopic assay (11) was run in 90 mM Trisacetate buffer, pH 7.75, at 37°C. Unreacted glc-6-P and product, MI-l-P, were hydrolyzed with calf intestinal phosphatase (P. L. Biochemicals, Milwaukee, Wise.) as described in the assay procedure. In a slight modification of that procedure, the entire product was separated by descending paper chromatography in methanol-88% formic acid-water, B&15:5 (v/v) for 12 h. The MI-containing region of the paper was cut out and immersed in 10 ml of toluene containing 0.55% Permablend III (Packard Instruments Co., Downers Grove, Ill.) for liquid scintillation analysis. To confirm complete hydrolysis of phosphate esters in the phosphatase step, an aliquot from the phosphatase reaction was separated by thin-layer chromatography on cellulose with ethyl acetate-pyridine-water, 10:6:5 (v/v). Regions corresponding to glc-6-P and glucose, as determined by accompanying standards, wer#e inspected for radioactivity. This test established the fact that all samples were completely hydrolyzed, including those from studies involving pH and inhibitor effects. The enzyme is stable when preincubated as much as 2.5 h in the presence and absence of substrates. Less than 1% of glc-6-P is consumed in this period, and the reaction is linear for at least 2.5 h. Therefore, velocity measured is initial velocity. The reaction rate is proportional to enzyme concentration. Isolation ofsynthase. Pollen (30 g) was ground in

200 ml of 50 mM Tris-acetate containing 1 mM GSH, pH 8.0, in a Dual1 glass-to-glass homogenizer (Kontes Glass Co., Vineland, N. J.) using 50 vertical strokes to complete the breakage of pollen grains. The suspension (homogenate) was centrifuged (18,OOOg, 30 min, 4’C). The supernatant (crude extract) was treated to recover a 35 to 55% ammonium sulfate precipitate which was redissolved in 20 ml of 50 mM Tris-acetate containing 1 mM GSH, pH 8.0. This was loaded on a column of Sephadex G-200 (2.5 X 90 cm) and eluted with 300 ml of GSH-less 50 mM Tris-acetate buffer. Synthase-containing fractions were pooled and adjusted to 60% saturation with ammonium sulfate to precipitate the enzyme which was recovered by centrifugation, and redissolved in 5 to 10 ml of 25 mM histidine-HCl or imidazole-HCl, pH 6.5. The solution was loaded on a chromatofocusing column of PBE 94 (0.9 X 35 em, Pharmacia Fine Chemicals, Piscataway, N. J.) and eluted with Polybuffer 74 adjusted to pH 4.5. Synthase eluted between pH 5.5 and 5.25. Protein was assayed with Coomassie blue (12). Reaction with thiol-reactive reagents. To study the protective effect of glc-6-P and/or NAD+ on synthase inhibition by the thiol-reactive reagents PCMB and NEM, enzyme was incubated with substrate(s) for 5 or 10 min at 32°C and then exposed to the thiolsensitive reagent. With PCMB as inhibitor, the reaction mixture was adjusted to pH 7.75, while with NEM it was adjusted to pH 7.0, a value more specific for reaction with sulfhydryl groups ((13), see p. 219). Subsequent assays of activity were made at pH 7.75 at 37’C.

TABLE

RESULTS

AND

DISCUSSION

Partial purification of synthase is summarized in Table I. Chromatofocusing provided 256-fold purification but overall yield was low; therefore, subsequent studies of pine pollen synthase utilized enzyme from the Sephadex G-200 step. A molecular

I

PARTIALPURIFICATIONOFPINEPOLLENSYNTHASE Protein (mg)

Treatment Homogenate* 35-50% Ammonium Sephadex G-260 Chromatofocus

sulfate

precipitate

4251 594 142 1

a One unit of enzyme is the amount needed to convert *From 30 g (dry pollen.

Total activity” (mUI 70 41 46 4.1 1 rmol of glc-6-P into MI-1-P/min.

Specific activity (mU/mg protein) 0.016 0.069 0.324 4.10

374

GUMBER,

LOEWUS,

weight of 155,000 + 10,000 was determined for synthase from this stage. This value is similar to that obtained for lily pollen synthase (157,000 + 15,000 Da), which has a subunit weight of 61,000 f 5000 Da (15). Attempts to incorporate a DEAE-cellulose step into the purification scheme (14) led to low yields and an unstable enzyme preparation. Moreover, attempts to purify the enzyme on PCMB-agarose affinity columns, as suggested by the observations reported herein, were unsuccessful. Pine pollen synthase exhibited a pH optimum between 7.25 and 7.75, slightly lower than the optimum (pH 7.8 to 8.5) of lily pollen (15), but within the broad range (pH 7.2-8.6) reported for synthase from other plant, fungal, and animal sources (7). The apparent K, = 0.33 mM for glc-6-P (NAD+, 1 mM) was greater than the true Km of 0.07 mM with lily pollen synthase (15). Increasing the concentration of Tris-acetate buffer from 10 to 70 mM tripled the enzyme reaction rate, which then remained unchanged up to 100 mM. Enzyme activity was measured in 90 mM Tris-acetate buffer. Ammonium ion stimulated the activity of pine pollen synthase just as reported for synthase from other sources (7). In this study, 5 mM ammonium acetate was included in each assay. The effect of pH (pH 6.5-9.0) on apparent Km and apparent V,,, of synthase when glc-6-P varied from 0.06 to 2.4 mM (NAD+, 1 mM) is given in Fig. 1 as a plot of log ( Vm,,/Km) against pH ((16), see p. 196). The plot suggests involvement in the reaction mechanism of two groups with pK,‘s of 7.6 and 8.6, possibly histidyl and sulfhydryl groups, respectively (17). Synthase from the 35 to 55% ammonium sulfate step was stable for at least 6 months at -20°C. Enzyme from the gel-filtered step lost activity when stored in 50 mM Tris-acetate, pH 8.0, under similar conditions. Addition of glycerol, NAD+, DTT, or bovine serum albumin failed to prevent this loss. Variable amounts of endogenous NAD+ remained bound to synthase during purification (see Ref. (7) for earlier citations). Pine pollen synthase retained about 80% of its NAD+ requirement after the first

AND

LOEWUS

FIG. 1. Plot of log (V,,,/K,,,) against pH for pine pollen synthase with glc-6-P as variable substrate and NAD+ held constant at 1 mM. Assays were made at 37°C in 50 mM Tris-acetate buffer.

salt precipitation. Subsequent steps of purification remove an indeterminant amount of bound NAD+. All assays included 1 mM NAD+ to assure saturation of the enzyme as regards this requirement. 2-Deoxy-glc-6-P, a strong inhibitor of synthase from other sources (15,18-20) at 0.1 mM inhibited pine pollen enzyme by 35% using the standard assay conditions. Inorganic phosphate and pyrophosphate, which are competitive inhibitors of Neurospwa synthase (21-24), both inhibited pine pollen synthase 70 and lOO%, respectively, at 10 mM inhibitor. The significance of inorganic phosphate inhibition as it relates to MI metabolism in pollen is worth noting. Phytate, a constituent of pine pollen (25), is hydrolyzed during germination by phytase (26) with concomitant release of inorganic phosphate. Conceivably, this release will inhibit synthase activity until endogenous sources of MI, both free and phytate derived, are utilized (7). MI-phosphatase (EC 3.1.3.25) (27), another source of inorganic phosphate, may also contribute to regulation of the synthase. None of the other products of MI metabolism tested (myo-inositol, D-c&o-inositol, L-chiro-inositol, myo-inosose-2, scyllo-inositol, deoxyscyllo-inositol, D-pinitol, and L-quebrachitol at 20 mM; or glucuronic acid l-phosphate and UDP-glucuronic acid at 10 mM)

SULFHYDRYL

INVOLVEMENT

IN myo-INOSITOL-1-P-SYNTHASE

375

inhibited the synthase. Lack of inhibition by these MI-related products suggests that inorganic plhosphate (and possibly pyrophosphate) is an attractive alternative for control df MI biosynthesis. Despite the limited information gathered thus far on gymnosperm synthase, comparison with angiosperm enzyme reveals different pH values (5.35 and 4.6, respectively) for elution after chromatofocusing, implying a difference in isoelectric points. This difference in net charge is also encountered in the elution behavior of the two enzymes from DEAE-cellulose (data not shown). Molecular and catalytic properties in whilch the two enzymes are similar include molecular weight, inhibition by deoxy-glc-6-P, stimulation by NH: ions, and the NA:D+ requirement (15). Reaction with Thiol-Reactive

Reagents

Earlier studies of synthase from yeast and rat mammary gland showed PCMB to be an inhibitor at
0

6

16 TIME. In,”

24

32

FIG. 2. Inhibition of 8% NAD+-independent synthase with NEM. Enzyme (1 ml, 0.4 mg protein), pretreated with 10 mM glc-6-P (O), 10 mM glc-6-P plus 4.8 mM NAD+ (A), or none (0), was treated with 1 mM NEM. Aliquots (200 ~1) were tested for residual activity.

Time

(mid

FIG. 3. Reactivation with 2-mercaptoethanol of PCMB-inactivated synthase. Enzyme (1.1 ml, 0.7 mg protein) was treated with 0.1 mM PCMB. At intervals, loo-p1 aliquots were removed for assay of residual activity (0). At 10 min (arrow), 600 ~1 of the reaction mixture was removed and combined with 4.8 mM 2mercaptoethanol. Aliquots (100 ~1) were removed at intervals to measure synthase activity (0). The activity of a control to which no PCMB was added is plotted before (A) and after (A) addition of 2-mercaptoethanol. The slight decrease in the control with time is probably experimental error since the enzyme does not lose activity up to 2.5 h.

and 3). The effect of preincubation with substrate on PCMB inhibition of pine pollen synthase is seen in Table II. Glc-6-P alone provided some protection against PCMB inhibition, more in the case of 70 than of 8% NAD+-independent enzyme. When the latter was preincubated with both NAD+ and glc-6-P, protection against PCMB inhibition was substantially greater. This suggests that the presence of both substrates was essential for protection, and the endogenously bound NAD+ explains the small protection with glc-6-P alone. Preincubation with NAD+ alone was ineffective in preventing inhibition. When NEM was added to 8% NAD+-independent synthase after pretreatment with glc-6-P alone or glc-6-P plus NAD+,

GUMBER,

376

LOEWUS, TABLE

AND

LOEWUS

II

PROTECTION BY SUBSTRATE AGAINST PCMB INHIBITION

OF PINE POLLEN SYNTHASE

Activity remaining (%)*

Experiment” (NAD+-independent activity, %)

Substrate(s) added to preincubation (mM)

1 min

10 min 0.6 2.1

1 (70%)

None

1.2

4.7

2 (8%)

NAD+ (4.2) Glc-6-P (10) None Glc-6-P (7.7) Glc-6-P (7.7) + NAD+ (4)

14.9

10.7

6.8 16.0 37.gc

2.0 3.4 21.0”

a A reaction mixture consisting of enzyme (0.8 mg protein in Experiment 1; 0.2 mg in Experiment 2), additions as noted in the table, and 20 mM Tris-acetate buffer, pH 7.75, was incubated for 10 min at 32°C. At the end of this period, PCMB (0.15 mM in Experiment 1; 0.05 mM in Experiment 2) was added (final volume, 1 ml). Aliquots (0.2 ml in Experiment 1; 0.15 ml in Experiment 2) were removed at intervals and assayed for activity. Enzyme, preincubated for the same period (10 min) in the absence of substrates and assayed at the same time intervals as PCMB-treated aiiquots, furnished the control. *Values are reported as a percentage of the uninhibited control. ‘During the lo-min period preceding addition of PCMB, about 5.5 X lo-’ pmol of MI-l-P was produced. The reaction contained about 3.3 X lo-” prnoi of synthase (molecular weight, estimated 1.6 X 10’). In view of the lack of reversibility of the synthase (4, 19), it is unlikely that the presence of this amount of product blocked PCMB binding.

only the latter provided significant protection (Fig. 2). These experimental observations lend support to other studies on the kinetics of lily pollen synthase, in which an ordered sequential addition of substrate, with binding of NAD+ preceding glc-6-P attachment, has been proposed (15). 2-Mercaptoethanol completely restored the enzymatic activity of PCMB-inactivated synthase (Fig. 3); evidence that the site undergoing attack at the active center is a sulfhydryl and that no other irreversible inactivation occurred. Although the specific role of this group in the interaction between synthase and substrates is unknown, one possibility is that of the base required for protonation-deprotonation of the substrate during the reaction (30). ACKNOWLEDGMENT We are most grateful sistance in this study.

to Brian

James for his as-

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SULFHYDRYL

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