THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol .261, No. Issue of April 5 , pp. 4481-4485,1986 Printed in U.S.A.

Polyphosphate Kinase from Propionibacterium shermanii DEMONSTRATION THAT THE SYNTHESIS AND UTILIZATION OF POLYPHOSPHATE IS BY A PROCESSIVE MECHANISM*

(Received for publication, September 3, 1985)

Nancy A. Robinson and Harland G. Wood From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

The mechanism of synthesis of inorganic polyphos- phate by polyphosphate kinase (EC 2.7.4.1) from Pro- pionibacterium shermanii is shown to be processive. Analysis of the synthesized polyphosphate on polyac- rylamide gels, which resolve on the basis of molecular weight, proves that the elongation reaction occurs without dissociation of intermediate sizes of the poly- mer from the enzyme. As a consequence, only high molecular weight polyphosphates are synthesized. The mechanism is processive both in the presence and ab- sence of basic protein. It has been shown previously that basic proteins stimulate the synthesis of polyphos- phate (Robinson, N. A., Goss, N. H., and Wood, H. G . (1984) Biochem. Int. 8, 757-769). In addition, using a similar method, it is shown that the reverse reaction, the utilization of polyphosphate to phosphorylate ADP, occurs by a processive mechanism. Accordingly, poly- phosphates formed by polyphosphate kinase in the cell would be entirely high molecular weight.

Polyphosphates (poly(P)’) are linear polymers of ortho- phosphate linked by phosphoanhydride bonds. These poly- mers have been detected in organisms encompassing bacteria and mammals (1). The in vivo function of poly(P) is uncertain; however, it has been suggested that they ( a ) act as a source of high energy phosphate; ( b ) are a phosphate reserve that can be mobilized under conditions of phosphate starvation; or ( c ) act as regulators of metabolic processes (1).

Two enzymes have been reported to catalyze the synthesis of poly(P) (1). The first 1,3-diphosphoglycerate-polyphos- phate phosphotransferase, which catalyzes Reaction 1 below, was first reported in extracts from Neurospora crassa by Kulaev and Bobyk (2).

1,3-Diphosphoglycerate + poly(P,) (1)

where n is the number of residues. This enzyme’s activity, however, was low or absent in other microorganisms tested (3) and was not detected in the propionic acid bacteria (4). Poly(P) kinase, which catalyzes Reaction 2, is the only other enzyme reported to catalyze in vitro synthesis of poly(P).

+ 3-phosphoglycerate + p~ly(P,+~)

ATP + poly(P.) + ADP + p0ly(Pn+1) (2)

This activity was first demonstrated with cell-free extracts

* This investigation was supported by Grant GM 29569 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: poly(P), a linear polyphosphate.

~ ~~

from yeast by Yoshida (5) and has been detected in a variety of bacteria including Escherichia coli (6), Salmonella minne- sota (7), Corynebacterium xerosis (8), Arthobacter atrocyanius (9), Propionibacterium shermanii (10, l l ) , Propionibacterium freudenrichii, and Propionibacterium arabinosum (4). All re- ports (6-9, 11) agree that the poly(P) synthesized by poly(P) kinase exhibits characteristics of long chain poly(P). There is no evidence of formation of short chain acid-soluble poly(P) intermediates (6,8,11). Kornberg et al. (6) suggested that the mechanism of synthesis of poly(P) might be analogous to nucleic acid synthesis in which low molecular weight inter- mediates rarely accumulate. This hypothesis has not previ- ously been critically tested.

We have found ( l l ) , using poly(P) kinase from P. sher- manii, that the activity is increased in the presence of basic proteins such as polylysine and histone. Basic protein causes the enzyme to precipitate, and the synthesized poly(P) re- mains noncovalently bound to the basic protein-enzyme com- plex. The nature of the stimulatory effect of basic proteins is not understood. An electrophoretic method of resolving poly(P) which differs by one phosphate residue (upper limit, 100 phosphoryl residues) on polyacrylamide gels was devel- oped. These gels provide a sensitive and accurate system for evaluation of the polymer composition of low molecular weight samples of poly(P). The enzymatically synthesized poly(P) was found to migrate on polyacrylamide gels as a species in excess of 200 phosphate residues.

We here show, using these gels and a procedure based upon the work of McClure and Chow (12) ‘with nucleotide polym- erases, that, in the presence or absence of basic protein, the mechanism of synthesis of poly(P) is processive and that the mechanism of utilization of poly(P) (Reaction 2 from right to left) is also processive.

EXPERIMENTAL PROCEDURES

Enzyme Preparation-A partially purified enzyme from P. sher- manii cells grown in either lactate or glycerol media (4) was obtained essentially as described (11) through the 40% ammonium sulfate fractionation step. This pellet was stored in saturated ammonium sulfate a t -20 “C until used. The following changes in the purification scheme increased the recovery of the enzyme 20-fold. A solution of 250 mM potassium phosphate, pH 7, containing 50 mM imidazole, 1 mM EDTA, and 0.7 mM 2-mercaptoethanol was used to elute poly(P) kinase from a Whatman P-11 cellulose column (5 X 38 cm). A linear gradient (0-500 mM) of NaCl in 10 mM potassium phosphate, pH 7, containing 1 mM EDTA and 0.7 mM 2-mercaptoethanol was used to elute the enzyme from a Whatman DE52 cellulose column (4 X 13 cm).

Poly(P) Kinase Assay-Poly(P) kinase activity was assayed by three methods: (a) a radioactive assay for poly(P) synthesis; ( b ) a spectrophotometric assay for poly(P) synthesis; and ( c ) a spectropho- tometric assay for poly(P) utilization.

The radioactive assay was performed with 0.5 mg/ml basic protein present as described (11) except that the concentration of [32P]ATP

4481

4482 The Mechanism of Polyphosphate Kinase

was increased to 0.8 mM. The specific activity, as measured in the polylysine-stimulated radioactive assay, was 1.25 units/mg of protein. Units are in pmollmin.

The spectrophotometric assay of synthesis involves regeneration of ATP using pyruvate kinase (ATP-regenerating system). The re- action mixture contains, in a final volume of 0.3 ml, 0.8 mM ATP, 20 mM potassium phosphate, pH 6.0, 10 mM MgC12, 2 mM phosphoen- olpyruvate, 0.5 mM NADH, 1 unit of pyruvate kinase, 1 unit of lactate dehydrogenase, and the poly(P) kinase. By this procedure the specific activity was 0.26 unit/mg. The reaction is monitored at 340 nm in a Gilford spectrophotometer. When 0.5 mg/ml polylysine is added to the assay, the rate of reaction is comparable to that measured with the radioactive assay. Since a polylysine-poly(P) kinase-poly(P) pre- cipitate forms ( l l ) , however, only the initial rate can be determined.

The reverse reaction, synthesis of ATP from poly(P) and ADP, is assayed spectrophotometrically. The reaction contains, in a final volume of 0.3 ml, 2 mM ADP, 2 mM MgC12,20 mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonate buffer, pH 8.0, 150 pg of Sigma type 75 plus poly(P) or the concentration of poly(P) indicated in the text, 7 mM glucose, 0.5 mM NADP, 1 unit of hexokinase, 1 unit of glucose- 6-phosphate dehydrogenase, and poly(P) kinase. The specific activity was 0.31 unit/mg. The reverse reaction is inhibited by basic proteins and at the 0-40% (NHI)2SOI stage of purification; 0.2 mM P',P5- di(adenosine-5')pentaphosphate is included in the assay to inhibit adenylate kinase (13).

Isolation of Enzymatically Synthesized Poly(P)-[32P]Poly(P) was isolated as described (11). Briefly, the [32P]poly(P)-protein complex was sedimented by centrifugation, and the complex was treated with proteinase K. The resulting mixture was extracted with phenol, and the poly(P), which remained in the aqueous phase, was concentrated by precipitation in 66% ethanol. This procedure separates the ["PI poly(P) from the excess [32P]ATP. When uhlabeled poly(P) was synthesized, it was done in the absence of basic proteins using the ATP-regenerating system described above. It was not necessary to remove the ATP since the poly(P) was visualized on gels by staining with toluidine blue and ATP does not stain with the dye. Centrifuga- tion and the proteinase K treatment were omitted, and the poly(P) was obtained directly from the reaction mixture after addition of EDTA (100 mM final concentration), by extraction with phenol and then precipitation in 66% ethanol.

Polyacrylamide Gel Electrophoresis and Determination of Poly(P) Chain Length-Samples were electrophoresed on Maxam and Gilbert (14) polyacrylamide gels and analyzed either by autoradiography or, for unlabeled poly(P), by staining with toluidine blue as described (11). The electrophoresis was carried out at 10 mA for 1.5-2.0 h until the dye, xylene cyanol, migrated 7 cm. The chain length of poly(P) up to 100 phosphate residues was determined by the counting method described previously (11). Sigma poly(P) types 5, 15, and 35, all of which are heterogeneous mixtures of poly(P), are electrophoresed into a 15% gel, lO:l, acrylamide to bisacrylamide. Chain length is assigned by counting the generated sizing ladder. Sigma tripolyphos- phate and tetrapolyphosphate are practically homogeneous and are used as reference points. In order to stain the lower molecular weight poly(P) species with toluidine blue, the gel is placed on processed x- ray film at 4 "C for 1 h. It is not known why this treatment causes the short chain poly(P) to stain.

Isolation of Poly(P) of Limited Size from Commercial Poly(P)- Sigma poly(P) type 35 was electrophoresed into a preparative (0.15 X 15 X 40 cm) 15% polyacrylamide gel at 50 mA until the dye, xylene cyanol, migrated 7 cm. Poly(P) was eluted from 1-cm wide horizontal strips of the gel with 0.5 M ammonium acetate, pH 6.5, the mixture filtered, and the poly(P) concentrated by ethanol precipitation (66% final concentration). The concentration of isolated poly(P) was de- termined using a toluidine blue dye-binding assay (15) with 2,4,6,8, and 10 pg of Sigma type 35 poly(P) as the standard.

Materials-Poly-L-lysine was from United States Biochemical Corp. (Cleveland, OH), [32P]ATP from ICN (Cleveland, OH), pro- teinase K from Boehringer Mannheim (Indianapolis, IN), 13255 cellulose thin layer chromatography plates and XAR-5 x-ray film from Eastman, 2 times crystallized acrylamide from Accurate Chem- ical and Scientific Corp. (Westbury, NY), N,N'-methylenebisac- rylamide from Bio-Rad, and calf thymus histone IIA, toluidine blue 0, PIP"-di(adenosine-5')pentaphosphate, polyphosphate glass types 5, 15, 35, and 65, tripolyphosphate, tetrapolyphosphate, hexokinase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and py- ruvate kinase (all sulfate free) were from Sigma. Reagent grade phenol was distilled before use. The poly(P) glucokinase was a partially

purified preparation from P. shermanii (16). All other chemicals were reagent grade.

RESULTS

Demonstration That the Mechanism of Poly(P) Synthesis Is Processive-A synthetic processive mechanism is defined as a process in which dissociation of the polymer from the enzyme does not occur after addition of each monomer. Thus, intermediates of chain length shorter than the end product are not observed. The mechanism of poly(P) k' mase was examined by analyzing the poly(P) synthesized in the pres- ence of histone on a polyacrylamide gel (Fig. 1). After a 15-s incubation (lane 2) and at all subsequent times, the synthe- sized poly(P) was entirely long chain. [32P]Poly P which was hydrolyzed in 1 N NaOH at 100 "C for 5 min was also isolated, and the results (lane 6) show that short chain poly(P) would have been detected if present. In addition, the possibility that short chained poly(P) was synthesized and present in the supernatant of the histone-poly(Pj pellet was examined. The supernatant was treated with charcoal to remove [3ZP]ATP and then analyzed on thin layer cellulose plates developed in 85% Ebel's solvent (17). At least 6 phosphate-containing species were observed including Pi, PPi, and ATP (data not shown). However, the same radioactive spots were found in controls in which the reaction was stopped before addition of poly(P) kinase or when [32P]ATP alone was analyzed. I t is concluded that short chain poly(P) is not formed by poly(P) kinase. These results show that the mechanism of synthesis is processive when histone is included in the assay.

A similar determination was undertaken in the absence of basic protein, but including the ATP-regenerating system, which permitted sufficient synthesis of poly(P) for the anal- ysis. Unlabeled ATP was used since the 32P of the ATP would have been diluted when regenerated from the P-enolpyruvate. The synthesized poly(P) was visualized with toluidine blue. The poly(P) synthesized in the absence of basic protein mi- grated solely as high molecular weight species (Fig. 2, lanes 3-6). Sigma poly(P) type 65 was subjected to the same isola- tion scheme, and all chain lengths were recovered with an approximate 50% yield (Fig. 2, lane 2). This control proved

1 2 3 4 5 6

- a0

- 35

FIG. 1. Evidence that in the presence of histone the mecha- nism of synthesis of poly(P) by poly(P) kinase is processive. Poly(P) was enzymatically synthesized using [32P]ATP, with histone present in the reaction, for the times indicated below. The ["PI poly(P) was isolated as described under "Experimental Procedures" and electrophoresed into a 15% polyacrylamide gel (201, acrylamide to bisacrylamide). An autoradiogram of the gel was made by an overnight exposure at -20 "C. Lanes 1-5 are with isolated pgly(P) synthesized for 0, 15, 30, 60, and 120 s, respectively. To prove that short chain poly(P) would be detected, a sample of [32P]poly(P) was hydrolyzed in 1 N NaOH at 100 "C for 5 min prior to isolation (lane 6 ) . The migration position of poly(P) of chain lengths 80 and 35 are indicated.

The Mechanism of Polyphosphate

1 2 3456 ad *

Kinase 4483

1 2 3 4 5 6 7 8 9

r w l

FIG. 2. Evidence that the mechanism of synthesis of poly(P) in the absence of basic protein is processive. Unlabeled poly(P) was synthesized for the time indicated, using the ATP-regenerating system, and the poly(P) was isolated as described under “Experimen- tal Procedures.” The isolated poly(P) was electrophoresed into a 10% polyacrylamide gel (101, acrylamide to bisacrylamide) at 10 mA for 2 h, and the gel was stained with toluidine blue. A control which contained 10 pg of poly(P65) was similarly isolated. Lane 1 is with 10 pg of poly(P65); lane 2, poly(P) isolated from 10 pg of pOly(P65); lanes 3-6, isolated poly(P) synthesized for 20, 15, 10, and 0 min, respec- tively. The spot to the left of lane I is the dye bromphenol blue, and the aberration in lanes 1 and 2 is due to a high concentration of salt. The migration position of poly(P) of chain length 40 is indicated.

that, if shorter chain Poly P had been synthesized, they would have been detected.

Comparison of Poly(P) Synthesized in the Presence or Ab- sence of Basic Protein and Tests of Their Purity with Poly(P) Gl~cokinase-[~~P]Poly(P) was synthesized in the absence of basic protein and also with 0.5 mg/ml histone or with 0.5 mg/ ml polylysine, then isolated and electrophoresed in parallel into a 10% polyacrylamide gel to directly compare the syn- thesized poly(P) (Fig. 3). After the synthesis, 0.5 mg/ml histone was added to the samples which did not contain basic protein. In contrast to experiments of Figs. 1 and 2, the poly(P) protein complex was precipitated with 5% trichloro- acetic acid prior to proteolysis. This procedure separates the poly(P) from unused [32P]ATP but does cause some hydrolysis of the poly(P) (compare Fig. 1, lanes 2-5 and Fig. 2, lanes 3- 5 with Fig. 3, lanes 2, 5, and 8). Nevertheless, it can be seen that the poly(P) synthesized either in the presence of histone or in the absence of basic protein appeared identical (lanes 2 and 5 ) . The poly(P) synthesized in the presence of polylysine (lane 8 ) appears to be more heterogeneous and of somewhat shorter length. The radioactive material is shown to be poly(P) since it was completely utilized by poly(P) glucokinase to phosphorylate glucose, forming glucose 6-phosphate (lanes 3, 6, and 9) .

Demonstration That the Mechanism of Utilization of Poly(P) Is Processiue-The mechanism of poly(P) utilization was investigated by examining the chain length of the residual unutilized poly(P) after incubation with poly(P) kinase. A fraction of poly(P) of limited size was isolated from commer- cial poly(P) as described under “Experimental Procedures’’ and used as a substrate to phosphorylate ADP. At the times indicated in the legend of Fig. 4A, aliquots of the assay mixture were added to EDTA (33 mM final concentration) and the samples extracted with phenol. The unutilizedpoly(P) was concentrated by precipitation with ethanol (66%) and

- 0 -

FIG. 3. Comparison of poly(P) synthesized in the absence of basic protein with that synthesized in the presence of either histone or polylysine. [32P]Poly(P) was synthesized in an assay containing the ATP-regenerating system. Poly(P) was isolated as described under “Experimental Procedures,” except that after the reaction was terminated by the addition of EDTA (33 mM final concentration)? 0.5 mg/ml histone was added to the samples which did not contain basic protein. Trichloroacetic acid, final concentta- tion 5%, was then added to all the samples, and the precipitate of poly(P) and protein was isolated by centrifugation. These precipitates were washed 2 times with 5% trichloroacetic acid and once with 100% ethanol. The remainder of the isolation scheme was identical to that previously described (11). Aliquots of the isolated poly(P) containing 50,000 cpm were incubated in 7 mM glucose, 7 mM MgC12,2 mM Tris, pH 7.5, 10 pg of poly(Pss) (added as carrier), and 11 milliunits of poly(P) glucokinase in a final volume of 20 pl. Samples were electro- phoresed into a 10% gel (lO:l, acrylamide to bisacrylamide). Shown is an autoradiogram of the gel exposed for 18 h. Lanes 1,2, and 3 are from the experiment in the absence of basic protein; 4,5, and 6 with histone and 7,8, and 9 with polylysine; 1,4, and 7 are zero time, 2,5, and 8 with isolated poly(P) synthesized for 20 min, and 3, 6, and 9 poly(P) synthesized for 20 min that had been incubated with poly(P) glucokinase. The radioactive spot in lunes 3, 6, and 9 is [32P]glucose 6-phosphate which was identified by co-migration with authentic glucose 6-phosphate on TLC cellulose plates developed in Bandurski and Axelrod’s basic solvent (18) (not shown).

analyzed on a polyacrylamide gel. As shown in Fig. 4A, the staining intensity of the poly(P) decreased with time, but the size of the remaining poly(P) was constant. Utilization of poly(P) without formation of shorter chain species proves that the mechanism of utilization is processive; a poly(P) molecule binds to the enzyme and does not dissociate until the polymer has been completely utilized.

Effect of Length of Poly(P) on Its Utilization-Two poly(P) fractions of different lengths were isolated, mixed, and used as the substrate for the phosphorylation of ADP. The residual poly(P), during the course of the reaction, was analyzed using polyacrylamide gels as above. The results (Fig. 4B) show that both sizes of poly(P) were used simultaneously.

DISCUSSION

We have proven that the mechanism of synthesis of poly(P) from ATP by poly(P) kinase is processive. Elongation of the polymer occurs without repetitive dissociation from the en- zyme, and, therefore, the poly(P) species that accumulate are of high molecular weight. This is true whether or not histone is added to the reaction (Figs. 1 and 2). This result indicates that, in the cell, all the poly(P) synthesized by poly(P) k’ mase would be high molecular weight. Clark et al. (19) have devel- oped a mild extraction procedure and have isolated poly(P) from P. shermanii grown in lactate medium. The poly(P) was exclusively high molecular weight. Their results support the theory that poly(P) kinase is responsible for the in vivo

4484 The Mechanism of Polyphosphate Kinase

A B

1 2 3 4 5 6 1 2 3 4 5

+>loo +

FIG. 4. Evidence that the mechanism of utilization of poly(P) is processive. A, poly(P), chain length longer than 100 phosphate residues and of a relatively uniform size, was isolated by elution from a polyacrylamide gel as described under Experimental Procedures.” 5 pgllane of this isolated poly(P) was initially added as substrate for the poly(P) kinase reaction. The reaction mixture was identical to the assay for utilization of poly(P) described under ”Experimental Procedures.” After 0-, 2.5, 5-, lo-, E-, and 20-min incubations (lanes 1-6, respectively), the remaining poly(P) was extracted with phenol, concentrated by precipitation in ethanol (final concentration, 66%), and analyzed by electrophoresis into a 15% polyacrylamide gel (201, acrylamide to bisacrylamide). The spot to the left of lane I is the dye bromphenol blue. B, two different molecular weight fractions of poly(P), one greater than 100 phosphate residues and the other having an average of 45 phosphate residues, were isolated as described under “Experimental Procedures,” mixed, and used as a substrate for the poly(P) kinase reaction. Unutilized poly(P) was isolated and analyzed as in panel A. 5 pg of each species were initially added per lane. Lanes 1-5 are with the poly(P) remaining after 0-, lo-, 15-,20-, and 25-min incubations, respectively. The spot at the right of lane 5 is the dye bromphenol blue.

synthesis of poly(P) in these bacteria. The stimulatory effect of basic protein is still a puzzling

aspect of this investigation. It was originally thought that the basic protein might “hold” the poly(P) at the active site of the enzyme and thus cause the formation of long chain poly(P), since it had been shown that a ternary complex between basic protein, enzyme, and poly(P) is formed (11). If this were true, it would be expected that when the basic protein was not present to bind the synthesized poly(P) to the enzyme complex, short chain poly(P) would be synthe- sized. However, as shown in Fig. 2, the mechanism remains processive in the absence of basic protein. Comparison of the poly(P) synthesized in the presence or absence of basic protein reveals that the poly(P) synthesized in the presence of poly- lysine is somewhat more heterogeneous and shorter in chain length than that made in the absence of basic protein or with histone added (Fig. 3). The significance of this result is not presently understood. Furthermore, the procedure developed by Clark et al. (19) to extract poly(P) from the propionic acid bacteria does not extract poly(P) precipitated with basic pro- tein. Apparently, either the simulation of poly(P) kinase by basic protein does not occur physiologically or, if it does, the poly(P) does not remain complexed to the basic protein.

Several different methods have been used to investigate the processivity of DNA polymerases. These methods include template challenge, product analyses, and cycling-time per- turbation (for discussion of methods, see Ref. 12). Since synthesis of poly(P) does not require a template, the only method applicable to the poly(P) kinase system was product

analysis. In investigations with DNA polymerases, the average number of nucleotides incorporated ( n ) before dissociation of the polymer from the enzyme is usually determined and has been found to be dependent upon temperature and ionic strength (20). We have so far been unable to obtain a precise measurement of n since the resolution of the polyacrylamide gels used in this investigation is limited to less than 100 phosphoryl residues. It is known that the poly(P) synthesized by poly(P) kinase has a higher migration position on polyac- rylamide gels than Maddrell salt (a preparation of high mo- lecular weight poly(P) commercially available from Sigma). NMR analysis, performed by Joyce Jentoft at Case Western Reserve University, and enzymatic analysis (21) of the aver- age chain length of Maddrell salt both yield a value of 450 f 20 phosphoryl residues. Thus, the value of n, under the conditions described in this communication, is greater than 450.

The mechanism of utilization of poly(P) to phosphorylate ADP was also shown to be processive by analysis of residual poly(P) on polyacrylamide gels (Fig. 4). The polymer, once bound to the enzyme, does not dissociate from it until com- pletely utilized. Thus, the poly(P) which was of fairly uniform size when initially added, decreased in concentration, but the size of the unutilized polymers remained constant during the reaction. If a mixture of two different sizes of poly(P) were added as substrates, both were used simultaneously (Fig. 4B). These results are in contrast to those obtained by Pepin and Wood (16) with poly(P) glucokinase from P. shermanii. This enzyme catalyzes the phosphorylation of glucose using poly(P) as the phosphate donor. The reaction occurs by a processive mechanism until the polymer is reduced to -100 phosphate residues, after which there is a switch to a prefer- ential nonprocessive mechanism. During the nonprocessive phase, the enzyme uses the longer poly(P) preferentially, and dissociation of the polymer occurs repetitively from the en- zyme. Thus the poly(P) decreases progressively in length during its utilization. If two sizes of poly(P), both less than -100 residues, are added as substrates, the longer size is used until it is approximately the length of the shorter species, and then both are utilized. Poly(P) kinase does not display such a preference (Fig. 4B).

The formation of a polymer by a processive process may be beneficial as compared to a nonprocessive process. In the nonprocessive process, small polymers accumulate whereas in the processive mechanism only long chain polymers are syn- thesized. The formation of large poly(P) without formation of intermediates may be advantageous since it avoids disrup- tion of the osmotic balance of the cell. In addition, a strictly processive process has potential advantage over a nonproces- sive process (20) in that the overall rate of reaction is depend- ent on the on and off rate of only one substrate rather than two, since the poly(P) remains bound to the enzyme until it reaches a large size.

The addition of inorganic phosphate or poly(P) is required for the synthesis catalyzed by poly(P) kinase? however, it is not known whether these compounds act as substrates (the initiator) or as allosteric regulators. We intend to examine this question in an effort to gain a better understanding of how poly(P) is synthesized and what regulates the synthesis.

Acknowledgments-We wish to thank Catherine Pepin who gen- erously donated the poly(P) glucokinase, Joan E. Clark who contrib- uted helpful discussion, and Helga Beegen who photographed and printed the figures.

N. A. Robinson and H. G. Wood, unpublished data.

The Mechanism of Polyphosphate Kinase 4485

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