Rivers Singleton, Jr.
Bioorganic Chemistry
Ames Research Center, NASA Moffett Field, Californio 94035
Phosphorus is one of the most important elements in biological systems. Yet, to many biochemists, the chemistry and reactions of this element are often poorly understood. The intent here is to review the chemistry of phosphorus and its biologically important compound, phosphate, with the goal of achieving a better understanding of the role this element can play in biological systems. The intent is not to present a current review of problems in phosphorus chemistry, but rather to organize and discuss material from organic chemistry, physical chemistry, and biochemistry in an attempt to arrive a t a unified picture of phosphorus involvement in hioorganic chemistry. Phosphorus is a group five element, located in the third period of the periodic tahle. Its physical atomic weight is 30.9840, its chemical atomic weight is 30.975, and its atomic number is 15. Seven isotopes have been reported, of which one, ::P, i s stable. The properties of the six radioactive isotopes are summarized in Tahle 1( I ) . The most Eommon experimentally used radioisotope, 32P, is easily produced via the reactions "p In + 3ZP Y (1)
-
Isotope
e
energy (meV)
11.00 3.94
86-
0.28 see 4.4 sec 2.6 min 14.3 da 25.0 da
1.78 1.28 2.24
R+
12.4 sec
6.1
2.1
Decay
hi?
Zap
8+
sup
zap
@+ @+
34P
a2P s3P
Y-
energy (meV)
Type of
3.3 1.71 0.25
... ...
Table 2. Comparison of Bond Angles of Some Phosphines and Amines
General Chemistry of Phosphorus
+ +
Table 1. Radioactive Isotopes of Phosphorus
+
+ :
3% in 3zP P (2) Reaction (2) is the preferred process for the preparation of carrier-free isotope (2). The isotope 32P is well suited for tracer studies. Its energy is sufficiently high that it can he counted by a variety of procedures, including the GeigerMueller and scintillation techniques. Yet it is not of sufficiently high energy to pose serious health hazards, if hnndled properly. The half-life is such that in many tracer experiments. corrections for decav are unnecessarv, vet it . . poies no long-term storage problems. For most applications, 33P should he as useful as SZP. I t is relatively G l d from an energetic viewpoint, having an energy approximately twice that of '4C. Its half-life is twice that of 32P, thereby making possible longer experiments without the necessity of decay corrections. The major restrictions on its use have been, first, the lack until recently of commercial availability, and second, the somewhat prohibitive cost. The electronic configuration of the neutral phosphorus atom is
Phosphine
Bond Angle
Amine
Band Angle
semiconductor properties of red phosphorus are consistent with a lareelv amorohous. hiehlv structure. - . ~olvmeric The varence elecironic configuration, 3s23p3, can lead to a wide variety of trivalent phosphorus compounds, of the general types: R,PH3-,; (RO).PR3-,; RXPC4-,; (RS),PR3.,; and (RzN),PR3.,. Since the p orbitals are a t right angles to each other, these compounds might he expected to have a pyramidal structure. However, as seen in Tahle 2 (3), the bond angles of many of these compounds show some deviation from that predicted for a pyramidal structure. In the case of the parent cobpound phosphine, the bond angles are close to the theoretical values, indicating a large measure of p-orbital bonding. In the other trivalent phosphorus compounds, however, some s-orbital participation must he involved, as can be seen by a comparison with the corresponding amines which are thought to involve essentially all sp3 bonds. Trivalent phosphorus compounds do not appear to involve the use of p, - p, bonding, as do some of the neighboring elements of phosphorus. This generalization is readily demonstrated by the series of heterocyclic compounds (3)
lsZ2s22pS3s'3p3 The 3p3 electrons can be used to form stable compounds, and it is these electrons which are involved in the bonding of elemental phosphorus. The element exists primarily in white and red forms. White phosphorus is metastable, has a high vapor pressure, and consis& of cubically packed tetrahedral P4 molecules. Above 900°C, this form undergoes a dissociation reaction to a dimeric molecule (3, 4). Red phosphorus, along with violet and various other metallic and semimetallic forms, is more stahle. The color and Adapted from lectures given by the author in the Bioorganic Mechanisms course, Department of Biochemistry, Case-Western Reserve University, Cleveland, Ohio during the author's tenure as a post-doctoralfellow of the American Cancer Society. 538
/ Journal of Chemical Education
The first two compounds are well documented in the chemical literature, whereas the last is unknown. Trivalent phosphorus compounds are hiphilic and can react as both nucleophilic and electrophilic centers (5). Bv analow -. with the amines. the nucleoohilic character probably arises from the remaining electron pair on the nhos~horusatom. The source of the electro~hiliccharacter is more complex. I t probably arises from a low electronic repulsion exerted towards a nucleophilic reagent due to the larger radius and more polarizable character of phosphorus (3). This dual reactivity is exemplified by the reaction
&pH
+
&'PC1
+
QP-PW
-+
HC1
(3)
Other phosphorus compounds include four, five, and six valent species. Trivalent compdunds can undergo nucleophilic attack, leading to quaternary phosphonium compounds having the general structure P(A)nC+, which involve only sp3 honding &P +' ROOH --t &P-OHRO &P=O ROB (4) These compoundsare often unstable and decompose to phosphoryl and related compounds. Phosphoryl and related compounds involve predominately sp3 honding, with additional p, - p, interaction. Since phosphorus compounds of biological importance have structures of this type, we will consider them in some detail. One can write the resonance structure (6) for a tetravalent phosphorus compound, using sp3 hyhridization only
-
+
a,e = APICAL b,c, d = EQUATORIAL
b,d = A P I C A L o,c,e. = EQUATORIAL
Figure 1 . Schematic reaction showing pseudorotation of pentavalent Species (8-10). Pseudorotatian is achieved by holding one equatorial point fixed ( c ) : the other two equatorial points (band d ) are stretched out from the plane of the page, while the two apical points ( a and e) are stretched back into the plane of the page.
also contributes to the rate enhancement. Phosphorus can form pentavalent species through extensive use of d-orbital interaction (3, 5). These species are all bipyramidal, and involve relatively weak honding. Some stable pentavalent species have been observed, such as the phosphoranes, which have been extensively studied by Ramirez (7)and others.
However, the remaining 2p electrons on the oxygen atom can now interact with the unfilled d,, and d,, orbitals of the phosphorus atom
This interaction can occur both with the d, .p , and d,, - p, orbitals, and results in a hond with triple hond character, being composed of one interaction and two a interactions. The remaining phosphorus d-orbitals can also interact to a limited extent with the other substituents on the central phosphorus atom. However, since these atoms lie outside the x-y-z planes, there is considerably less overlap. Based on these considerations, the phosphoryl structure can best he described by the resonance series
X X X From this series, it can he seen that an increase in the polarity of the P = 0 bond would facilitate attack on the phosphorus by increasing electrostatic interaction between the central phosphorus atom and the attacking nucleophile (6). This effect is demonstrated in the ester hydrolysis series below 0
2'
I
0
It is now believed that species of this type are formed as transients in the hydrolysis of cyclic phosphate esters, which undergo reaction millions of times faster than the co~espondingnon-cyclic esters (8-13). A key finding in this study was the observation by Westheimer (9) that hydrolysis of ethylene phosphate is accompanied by exchange with solvent water 0
HO-CH,-CH,-O-P-O-H
II
IS
I
Furthermore, the hydrolysis of methyl ethylene phosphate is accompanied by rapid hydrolytic cleavage of the methoxy group; this reaction is 106 times faster than the hydrolysis of trimethyl phosphate. The question that arises is how a strain effect could accelerate hydrolysis without causing ring opening. Westheimer (9) proposed that the reaction mechanism involves the formation of a pentavalent phosphorus intermediate, as shown in reaction (8).
I
OEt h,' = L2 X lO-'secC'
v
OEt k,'=6.5 X
VI
10-"set-'
OEt k< =7.8 X 10-6sec-'
VII
Since oxygen is more electronegative than sulfur, it tends to polarize the P = 0 hond to a greater extent, therehy increasing the attack on phosphorus (VI versus V). In the ester linkage (P-O-X), oxygen can conjugate more effectively with phosphorus, therehy reducing its electron demand. Sulfur does not do this as readily; one therefore sees an increased rate of hydrolysis with VII compared to VI. We should also note that in this case, the mercaptide ion is a better leaving group than the alkoxide ion, which Volume 50, Number 8, August 7973
/ .539
I
CHa
The following generalizations can be made regarding the reactions of these pentavalent phosphorus species (810). First, the two apical positions are weaker and more reactive than the three equatorial positions. A group attacking apically must also leave apically. Second, the preference mles dictate that positively-charged groups (e.g., O+Hzr prefer apical positions, negatively-charged groups (e.g., 0 - ) prefer equatorial positions, and neutral groups (e.g., OH or OR) can assume either position. Third, the position assumed in the bipyramid by any group is under steric control; for example, in reaction (81, the five-membered ring must assume one apical and one equatorial position in hoth rotation forms. Finally, as shown in Figure 1, the position of any group in the bipyramid can be changed through pseudorotation. It can be seen that this mechanism provides an explanation for the findings previously described. The H P 0 exchange arises from an apical attack of Hz180, pseudorotation and proton loss, followed by protonation of the 0in the apical position to HzO, and its subsequent loss. With hoth the methoxy and the ring oxygen in the apical position, hydrolysis can proceed with either the loss of the methoxy group or opening of the ring. Hexavalent comnounds of . ~ h o. s ~ h o r are u s relativelv stable and involve extensive d-orbital interactions (3).They have octahedral structures. As these comnounds play no role in biological systems, they will not be discussed-further. With this brief review of the types of reactions phosphorus can undergo, let us turn our attention to the reactions of the phosphate esters, since these are the phosphorus compounds of greatest biological importance. Hydrolysis of Phosphate Triesters
Under alkaline conditions, phosphate triesters undergo rapid hydrolysis to the diesters, followed by a slower reaction to other species (6). For the hydrolysis of trimethyl phosphate, the model compound for this series, the following observations have been made (1) the reaction is tint order with respect to the hydroxide ion
and the ester;
(2) the reaction involves entirely P - 0 bond cleavage; and (3) there is no exchange between the P-0 bond and water.
Points (1) and (2) suggest a nucleophilic attack of the hydroxide ion on the central phosphorus atom. Point (3) suggests the lack of formation of an intermediate which would be able to exchange with water. The mechanism which best fits these criteria is a concerted, nucleophilic attack of the hydroxyl ion on the phosphorus, with a concomitant displacement of the methoxide leaving group, as shown below 0
II
CH,-0-P-0-CH,
I
+
'OH
0 I
CHz 540
/ Journal of Chemical Education
*
The alternative to this mechanism is a two-step process in which the breakdown of the intermediate is too rapid to allow exchange; this reaction is experimentally indistinguishable from that described above. Hydrolysis of Phosphate Diesters
The non-cyclic disuhstituted phosphate esters are the most stable of the phosphate esters (6, 11, 12). as c a n readily be seen by comparing the first order rate constants of the monoanion of mono- and dimethyl phosphate
Since the diesters have an ionizable group with a p K of around 1.5, two reactive species are potentially possible. In stronelv -. alkaline solutions. the hvdrolvsis . . of dimethvl phosphate is first order with respect to the concentrations of the hvdroxide ion and ester: onlv 10% of the hvdrolvsis involves-a P - 0 bond cleavage. ~ h & ,the primary mechanism must involve a nucleophilic attack on the carbon, with a small percentage of attack occurring at the phosphorus. However, the extent of attack a t carbon depends on the leaving group present in the diester. The better the leaving group, the more attack occurs a t phosphorus. This reasoning has been the basis of the elegant synthetic pathways used by Khorana and others in the synthesis of polynucleotides (14, 15). Under acidic conditions, the disubstituted esters also appear to he hydrolyzed via two mechanisms. Some confusion seems to exist as to the exact amount of P - 0 cleavage, but values of 20-30% have been reported (6, 11). Thus there a m e a r to be at least two mechanisms aonlicable for the hy&olysis of disubstituted phosphate esters. There is a marked exception to the observation that the diesters of phosphate are stable. If a hydroxyl or other nucleophile is present vicinal to the ester, the reactivity is markedly increased. One example of this effect is the observation that methyl-2-methoxy ethyl phosphate is hydrolyzed less rapidly than is methyl-2-hydroxy ethyl phosphate; another example is the observation that the conversion of @-glycerolphosphate to the a-form does not involve an exchange with inorganic phosphate (16). In 1952, Brown and Todd (17) studied an extensive series of reactions and postulated that these reactions proceed via a five-membered ring system which undergoes spontaneous hydrolysis. As these reactions play an important role in biological systems, we will return to a discussion of them at a later point.
The highly unstable metaphosphate intermediate is hypothetical and must be considered a very transient species a t best. The above equation describing the hydrolysis of the monester involves a neutral species term. The involvement of this species has been conveniently studied a t increased ionic strength, due to the large positive salt effect (e.42~).The hydrolysis proceeds mainly with C-0 bond cleavage; however, some P-0 cleavage is observed. Furthermore, the rate is proportional to the Hammett acidity coefficient, Ho. From the Hammett-Zucker hypothesis, t h i s observation implies that the reaction is bimolecular and involves water in the rate-determining step. Two mechanisms can be formulated on the basis of these data
X
/ I 0
/
/
- EXPTL
-- CALC
I
1
2
3
4
5
6
7
8
Ham4
PH Figure 2. Hydrolysis of monomethyl phosphate at 100.1" (from Bunton, et al. ( 1 8 ) ;used with permission of the authors and publisher).
1
I
I
J
I
H H O H
Hydrolysis of Phosphate Monoesters
The monosubstituted phosphate esters have two ioniza'ble groups with pK's of around 1.0 and 6.5 (6, 11, 12, 18, 19). Thus, there are three potentially reactive species
,A pH profile for the hydrolysis of methyl phosphate is shown in Figure 2. The calculated line was determined in part by the equation
where M is the concentration of the monoanion; N is the concentration of the neutral species; and e.42~is the salt effect term. The lack of fit in the acid region is due to the fact that only the first term was used to calculate the line. The agreement improves if the neutral species terms are included in the calculation. These data and other kinetic data suggest that the rate-determining step in the reaction of the monoanion must involve the monoanion and water. An alternative mechanism might involve the neutral species and the hydroxide ion, a mechanism kinetically indistinguishable from that described above. However, this possibility can be eliminated by calculating the specific rate constant for the reaction of the neutral ,species with the hydroxide ion and comparing this constant with the observed second order rate constant for the hydrolysis of trimethyl phosphate (a neutral species by necessity) by the hydroxide ion (18). In the hydrolysis of almost all monoanions of singly substituted esters, P-0 bond cleavage has been almost exclusively observed. The generally accepted mechanism for this reaction is now as follows (12)
The hydrolysis of the dianion does not appear to proceed very readily, except in a few cases. One example of a reactive dianion is the hydrolysis of acetyl phosphate, which seems to he facilitated hy having a strongly anionic leaving group (20,21). The hydrolysis of glucose-6-phosphate is interesting in that it demonstrates a marked involvement of the dianion in hydrolysis (12). At p H values below 4, glucose-6-phosphate behaves like most alkyl phosphates. Above pH 4, however, the rate increases, showing that the dianion is more reactive than the monoanion. It appears that this increased reactivity arises from the relatively acidic hydroxyl group on C-1 donating a proton to the C-6 oxygen, allowing ready P-0 bond fission. The kinetic parameters for the hydrolysis of monomethyl phosphate are summarized in Table 3 (181, and demonstrate the variety of kinetic mechanisms involved in its hydrolysis. Other Mechanisms in Phosphate Ester Hydrolysis
Thus far, we have discussed the catalysis of phosphate ester hydrolysis mostly in terms of general acid and general base catalysis. Let us now turn our attention to other Table 3. Kinetic Parameters for the Hydrolysis of Monomethyl Phosphate
Form Monoanion Neutral Conjugate acid Conjugate acid
Bond
Rate Constant X
lo8 (100")
cis.
'
8.23 (s -I) 0.50 (9r1) 2.W (1 mole-%-') 1.08 (1 mole-'sr')
,,,
P-0 C-0 C-0 P-0
Molecu- salt
larity
Effect
? 2
nil
2
2
'
large + nil nil
From Bunton, et al. (18);used with permissionof the authors and publisher.
Volume 50, Number& August 1973
/
541
c1-c2
I I 0 O-H I o=p\ I
O-R
O\H
14 t4 11
+
+
C~-C2
n-6
phate is hydrolyzed by lutidine; however, in the presence of CaZ+, the rate is increased by another 1000 times (in the presence of 0.02 M CaZ+ and 0.2 M lutidine, the rate is 106 times the non-catalyzed rate) (22). Lowenstein has studied the non-enzymatic hydrolysis of adenosine triphosphate (ATP) by CaZ+, CdZ+, and Mn2+; these elements also catalyze a transphosphorylation of ATP with phosphate or carhoxylate ions. With Mn2+ an optimum occurs a t a ratio of MnZ+/ATP of 0.6 to 1.0. The rate is considerably slower than most enzymatic processes, hut may still he a valid model system for an enzymatic reaction. It has been suggested that the reaction proceeds via reaction (13) (23) (N = nucleoside, Me = metal ion)
6I
R-OH
o=v-00
I
OH
Figure3. Intramal~ularcatalysis by vicinal nucleophiles
sources of catalysis of hydrolytic reactions of these compounds (22). Phosphate esters can readily undergo hydrolysis catalyzed by various metal ions. There is a rather extensive literature in this area; in 1964, Cox and Ramsey (6) stated that there were over fifty references t o metal ion catalysis and since that review, the literature has been continually growing. In general, one can consider that the phosphate ester is coordinated by the metal ion, thereby facilitating attack on the central phosphorus atom. An example of this effect is the hydrolysis of a-glycerol phosphate, catalyzed by the hydroxides of lanthanum, cesium, and thorium a t alkaline DH.At DH 8.5. lanthanum hvdroxide accelerates the alka-
line hydrolysis of glycerol phosphate by 1000 times. It has been speculated that the mechanism involves the formation of a lanthanum complex between the hydroxyls of glycerol, phosphate, and water. Tetrahenzyl pyrophos542
/
Journal of Chemical Education
An interesting aspect of this proposed mechanism is not just the chelation of the terminal phosphate of ATP, hut the use of the metal ion to bring both reactants into close proximity for reaction. Another source of catalysis of phosphate esters is intramolecular catalysis by neighboring groups, as was previously discussed. As has been noted, vicinal nucleophiles can cause a rate enhancement of hydrolysis; however, no mechanism for this catalysis was discussed. According to Bruice and Benkovic (121, this rate enhancement arises from the generation of a highly reactive, pentavalent cyclic intermediate, as seen in Figure 3. A biological example. of rate enhancement via this mechanism is the hydrolysis of RNA under alkaline conditions. The reaction proceeds mechanistically as follows
-
Todd and coworkers (16) were able to synthesize the cyclic phosphate intermediates and t o demonstrate that they were hydrolyzed sufficiently rapidly to fit the proposed reaction mechanism. Furthermore, the cyclic intermediates have been trapped and characterized. This mechanism provides a rationale for the evolutionary selection of the deoxy sugars as the basis of the nucleic acid depository of genetic information (DNA). Since this molecule must have a high degree of chemical stability, evolutionary pressure has selected for the removal of the "destabilizing" hydroxyl group from its vicinal posi-tion. Thus far we have made little mention of enzymes as catalytic agents in phosphate systems. Let us now consider these catalysts and examine some enzyme-catalyzed reactions in terms of the phosphate chemistry already discussed.
Figure 4. First step in the proposed mechanism of action of ribonucle-. ase. The shaded areas represent binding sites for the two nucleotide bases. Sea t e n far details (from Blow and Streitz (26);used with permission of the authors and publisher).
Rihonuclease (RNase) is a classical example of rate enhancements arising from the generation of cyclic intermediates. This enzyme catalyzes the hydrolysis of ribonucleic acids resulting in a series of oligonucleotides having pyrimidine-3-uhosphates as terminus. Evidence has accumulated demoktr&ing that the reaction involves the formation of 2',3'-cyclic phosphates as intermediates in the reaction. Based on the currently available X-ray diffraction pictures and on other data (24-26), the mechanism outlined in Figure 4 has been proposed to describe best the mechanism of reaction of the enzyme. The imidazole of His-12 acts as a general base by picking u p a proton from the 2'-OH and facilitating its attack on the central phosphorus atom. At the same time, His-119 acts as a general acid, polarizing the phosphoryl bond, facilitating attack on the phosphorus, thereby breaking the 3',5' bond and generating the cyclic intermediate. The imidazoles of His-119 and His-12 then change roles; His-119 acts as a general base, facilitating the attack of water on the phosphorus, while His-12 acts as a general acid, donating a proton to the developing 2'-OH group. Although a pentavalent species is not shown, the reaction mechanism could very well he written involvingsuch an intermediate. Alkaline phosphatases are a broad class of enzymes which cleave phosphate from phosphorylated organic compounds a t relatively alkaline pH's (27). They have been isolated from a wide variety of sources and have a wide range of svecificities. Thev have pH maxima . eenerallv .. around 9-10 and often have metal cofaccors. 'l'he enzvme from Ewharichia coli is a mod example of the type. it has been demonstrated to involve a reactive serine a t the active site. The reaction apparently proceeds in two steps: (1) an initial phosphorylation of the enzyme with the loss of the alcohol; and (2) hydrolysis of the phosphorylated enzyme by water. Schwartz (28) has proposed the following mechanism of action
The role of the metal is most probably to neutralize the high negative charge on the dianion, thereby facilitating attack of the serine hydroxyl. One very interesting aspect of this enzyme is its ready phosphorylation by inorganic phosphate, hut only at relatively acid p H values. The p H maximum of the enzyme phosphorylation coincides with the point of maximum concentration of the monoanion and falls off rapidly with increasing pH (28). A very important function of phosphate in biological systems is the stabilization and activation of low molecular weight compounds for use in biosynthetic reactions. There are numerous examples of this function in many biological reactions (29), so we will cite only a few examples. In carbamyl phosphate, phosphate facilitates attack of nucleophilic groups on the carhamyl moiety
1I I
coo'
H-0-P-0'
0
I o=c I
NH,
+
I
HsN--CH
-+
I
CK
I coo0
Reactions of this type are very important in the urea cycle enzymes and in the hiosynthesis of the pyrimidines. There are numerous other examples of this type of phosphate involvement in biological systems. In the hiosynthesis of isoprenoids leading to steroids, pyrophasphate activates the carbon atom alpha to a double bond
ROH
+
Volume 50, Number 8, August 1973
/
543
This reaction is further facilitated by the hydrolysis of the product pyrophosphate by pyrophosphatase, thereby pulling the reaction to the right. This effect is frequently found in many biosynthetic pathways and often allows a reaction with an unfavorable equilibrium constant to proceed. .- -~~ In phosphoenolpyruvate (PEP), phosphate serves to stabilize the highly reactive en01 species for condensation reactions. A classical reaction of this nature is the carboxylation of P E P to yield oxaloacetate (30)
behavior of phosphate in chemical systems. It is interesting to note that in many respects, these principles resemble those governing the mechanistic behavior of carbon. Finally, we have hopefully shown that most hiological reactions of phosphate behave according to these mechanistic rules. Acknowledgment
I thank my Ames colleagues, especially Dm. R. D. MacElroy, L. I. Hochstein, and M. R. Heinrich, for reviewing and commenting on the manuscript. I also thank Dr. Gerald D. Weatherby of Lake Superior State College, Saulte Ste. Marie, Michigan for his critical reading of the manuscript. I thank the National Academy of Sciences/Nationa1 Research Council for their award of a Research Associateship. Literature Cited
Three enzymes catalyzing this reaction include: (1) PEP carhoxylase (where R = OH); (2) PEP carboxytransphosphorylase (where R = Pi); and (3) PEP carboxykinase (where R = ADP). Phosphoenolpyruvate can also condense with other nucleophiles (e.g., erythrose-4-phosphate in the shikimic acid pathway) in analogous biosynthetic reactions. It is obvious that the list of examples of this role of phosphate in biological systems could grow to he quite large. The examples cited will hopefully serve as a guide to other reactions. In summary, the chemistry of phosphorus and especially phosphate is relatively well understood. Basic principles have been established describing the mechanistic
544
1 Journal of Chemical Education
I , ~ m h H . L . m ~ . n d h c . k of C h p m ~ r yand P h ) . . n " ~ m h cd .&,tor Wmr. R V Vhcm#calHubhri,,. C I ~ < ~ l n n1 Sd h . p 1I.i ? W d r m R .I Fa6 r TUPR a d m c h m ~ 8 .hlanual ' 2nd rd I. I hr Hadm'hrm.. cal Centre. Amersham, 1966, p. 49. (31 Hudson. R. F., "Struefure and Mechanism in 0rgano.phosphorus Chemistry;' AcademicPress,NewYork, 1 9 6 5 , ~1. . (4) Van Wszer, J. R.,"Phosphorus and its C o m ~ u n d s . "Vol 1, hterjcienco, New York. 1 9 5 8 , ~I.. (9 Kirby, A. J., and wanen, S. G., "The ~ ~ nChemistry i e of Phosphorus." Elsw~ e rAmstoldam. , 1967, p. 1. (61 Cox, Jr., J.R., andRamsay, O.B., Chsm. Rsus., 64,317 (LWI. (71 Ramirer,F.,Aeeounts C h m . Rea. 1,168i1968). (8) Misloru,K.,ArountsCham. Rw., 3.321(19701. (91 Westheimer, F. H., Acrounta Chem Rex. I, 70 (19BSI. (101 Hudson,R. F.,sndBroun, C.,AecounfsChem. Rea, 5, W4(19721. (111 Bunton, C.A.,Accounfs Chem Re& 3,257i19701. (12) Bruice, T., end Benkauic. S., "Bimrganic Meehanhms,' Vol. 2. W. A. Benjamin, NewYark, 1966.P. 1. (131 Donnis,E.A.,and Westheimer,F. H., JAmer. Chem. Sor., 88.3432(1%61. (141 Khoxans,H. G., Fed. h c . , 19,931119M)1. (15) Kha1ana.H. G..Rioehm. J , 109.709i19681. (161 Broun, D. M., and Todd, A. a,. in ''The Nudeic Acids: Vol. 1 (Editors: Chargaff, E., and Dsvidson, J. N.), Aeademiehss, NewYark, 1955, p. 409. (17) Brnum.D.M., endTadd,A.R., J Chem Soe., 52,11952). (18) Bunton, C. A,. Lleweilyn, D. R., Oldham. K. G., and Vemon, C. A., J . C h m . Soe.. 3574 (1958). (191 Bunto", C. A,. Lleweilyn. D. R.. Oldham. K. G., and vemoo, C. A., J C h m Soe., 3588(19581. (20) Desahata, G.,andJandu, W.P., J A m e r Chem. S e t , 83,44M)(19611. (211 JencLs, W. P., Bmakhavan Sympos. Biol., 15,134 (1962). (221 Bender, M. L., and Bresloru, R., in "Comprehensive Biochemistry," Val. 2. (Editors: Florkin, M., and Sfotz. E. H.1. Elrvier, Amsterdam, 1962, p. 1. (231 Ialuenstein. J. M..Biochem. J., 70.222 (19581. (24) Findlay, D., Herrips, D. G., Mathias, A. P.. Rahio, B. R., and h s , C. A . Biocham J.. 85. 152(19621. (251 Deavin,A.,Mathiss,A. P., andRabin, B, R.,Notve. 211,252(19661. (26) Blow, D. M.. and Steitz,T. A.,Ann. Re". Biochm.. 39,9111970). I271 Reid. T. W.. and Wilson. I. B.. in "The Enzvmes." . Vol. 4 (3rd 4.1. .. (Editor . B&, P. D.) ~ ~ N~e r u ~ ~ . dkIWI, , p. 373. ~ ~ i ~ (281 Sehwanz. J . H . , h r . Not. Acod Sei. (U.S.A.l,49.871 (1963). (29) Mahior. H. R.. and Cords. E. H.. "Biological C h o m i s W (2nd ed.1, Harper and Row. NevYork, 1971, p. 377. (W) Utter, M. F., and Kolonhrsnder, H. M., in "The Enzymes," Vol. 6 (3rd ed.1 (Edifor:B~er,P.D.),AcademicPnss,NewYnk, 1971.p. 117.
. .
Plees,
.
