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Thermal enzymes. VIII. Properties of a heat-stable inorganic pyrophosphatase
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
60,
439-451
(1956)
Thermal Enzymes. VIII. Properties of a Heat-Stable Inorganic Pyrophosphatasel Connell Marsh2 and Walter From the Department
of Chemistry,
University
Received February
Militzer
of Nebraska, Lincoln,
Nebraska
7, 1955
Soluble fractions from specific cell granules of a thermophilic bacterium, (NCA No. 2184), have been shown to contain apyrase and adenosinetriphosphatase (ATPase) (1, 2). During the course of experiments with the ATPase, a highly active inorganic pyrophosphatase (PPase) was found to be intimately associated with this enzyme. The PPase is activated by magnesium and, in general, resembles the inorganic PPase from yeast (3, 4) in its catalytic properties. The resistance of the thermophile enzyme, however, to denaturation by heat is in marked contrast to the heat lability of the yeast enzyme. The thermophile PPase is stable for almost an hour at 75°C. while the yeast PPase is rapidly inactivated at temperatures above 40°C. The kinetic and thermal data obtained by Kunitz for the crystalline yeast enzyme provided st,andards of comparison for the various properties of the PPase from our thermophile. This communication is devoted to a report on some of the properties of the thermal PPase, including metal-ion activation, enzyme-substrate behavior, effect of pH on activity, and thermal
and kinetic data. Values for AR”, AHt, A#, and E have been calculated from thermalinact.ivation data and, wherever possible, compared to values calculated from
Kunit.z’
data
(3).
1 Aided by a grant from the Division of Research Grants and Fellowships, U. S. Public Health Service, National Institutes of Health. 2 Present address: Department of Animal Pathology and Hygiene, College of .1griculture, University of Nebraska, Lincoln, Nebraska; 439
440
CONNELL
MARSH AND WALTER
MILITZER
EXPERIMENTAL
Preparation of PPase Growth and harvest of the thermophiles were conducted as described in a previous paper of ,this series (5). The preparation of granule suspensions, and the extraction and partial purification of PPase was essentially the same as reported earlier for the ATPase (1,6). For extraction of the PPase, the granule suspensions containing approximately 40 mg. protein/ml. were allowed to age in the refrigerator for about a week before fractionation. This procedure gave a better yield of enzyme. Similar to ATPase, the PPase activity was found in the precipitate from both the 0.35 and 0.5 saturated ammonium sulfate fractions following preliminary separations with acetone. The 0.35 fraction was less highly colored than the 0.5 fraction and usually contained less protein. The best PPase preparations gave Q,, values of from 15,000 to 25,060 and contained from 3 to 5 mg. protein/ml. The PPase was found to be inactivated by freezing and thawing but remained stable for months if kept at refrigerator temperatures without freezing.
Determination of Enzyme Activity Enzymatic activity was determined by measuring the amount of orthophosphorus liberated, using the calorimetric method of Fiske and SubbaRow (7). Standard PPase incubation mixtures contained 1.0 ml. of pH 7.5 borate buffer, 0.3 ml. of 0.032 M sodium pyrophosphate, 0.3 ml. of 0.015 M magnesium chloride (or magnesium sulfate), 0.1 ml. enzyme extract, and sufficient glass-distilled water to make a final volume of 1.8 ml. The mixtures were incubated at 60°C. for 15 min., after which the tubes were immersed in ice water, and 0.2 ml. of 5Oyo trichloroacetic acid was added to stop the reaction and to precipitate the protein. The precipitate was removed by filtration, and suitable aliquots were pipetted into 25-ml. volumetric flasks for the colorimetric determinations. Duplicate controls were included in each set of determinations. The enzyme in the controls was inactivated by immersing the tubes in boiling water for 5 min.
Procedure for Thermal Inactivation Studies One-tenth milliliter aliquots of the enzyme preparation were pipetted into thin-walled 18 X 150 mm. Pyrex tubes containing 1.0 ml. of 0.067 M borate buffer, pH 7.5, and 0.1 ml. of distilled water, giving a final protein concentration of 0.25 mg./ml. At zero time the tubes were immersed in a constant-temperature water bath, two tubes being reserved for determination of the activity of the unheated enzyme. After appropriate time intervals the tubes were withdrawn and cooled in an ice bath. To each of the tubes were added 0.3 ml. of 0.032 M sodium pyrophosphate and 0.3 ml. of 0.02 M magnesium sulfate, after which they were incubated for 15 min. at 60°C. The difference between the zero-time activity and the activity after heating was used as the measure of inactivation of the enzyme.
THERMAL
ENZYMES. TABLE
E$ect
of Various
Ions
upon
VIII
441
I
Activity
of Pyrophosphntase
Incubation mixtures contained 1.0 ml. borate buffer, pH 7.5; 0.3 ml. sodium pyrophosphate, 0.032 M; 0.1 ml. enzyme solution; 0.1 ml. distilled water; and 0.3 ml. activating ion solution. Activit,ies are expressed as micrograms P released in 15 min. at 60°C. Activating
hIgSO*
(0.02
ion
.ktivity
M)
595
CoClz (0.02 M) hfnSOl (0.02 M) CaC12 (0.02 M] RESULTS
110 0 0 AND DISCUSSION
General The PPase showed a positive requirement for magnesium as an activating ion. There was no measurable activity where magnesium was omitted from the incubation mixtures. There was some activation by cobalt, but none by manganese or calcium. Table I shows the relative efficiency of these ions as activators. Crystalline PPase isolated by Kunitz from yeast (3) was activated best by magnesium and was also activated by cobalt and manganese, while calcium was inhibitory. Specificity Most of the PPase preparations also contained ATPase, and some would hydrolyze sodium tripolyphosphate slightly. There was no h;vdrolytic action on sodium trimetaphosphate and no phosphomonoesterase activity. Swanson (S), Zeller (9), and Krishnan (10) have suggested that in view of the marked similarities of ATPases and PPases, which are activated by magnesium, these enzymes may possibly be one and thr same ent,ity, at least in some instances. Krishnan obtained essentially constant PPase-apyrase activity ratios at various stages of purification of extracts from Penicillium chrysogenum. He concluded that one enzyme was responsible for t’he hydrolysis of both adenosine triphosphatc (ATP) and pyrophosphate. Similar studies with the thermophile e11zymes showed no such correlation of activities. Some fractions VOW tained ATPase and no PPase, and vice versa, while other fractions caontainedboth activities. Furt’hermore, preparations whirh initially con-
442
CONNELL
MARSH
AND
WALTER
FIG. 1. Effect of pH on enzyme activity;
MILITZER
Verona1 buffer, 0.10 M.
tained both ATPase and PPase rapidly lost their PPase activity upon repeated freezing and thawing, or during storage in the frozen state. The thermophile enzymes thus appear to be distinctly separate entities. The pH-activity curve for the thermal enzyme (Fig. 1) is broad and flat, somewhat like the curves obtained by Krishnan (10). Kunitz obtained rather broad pH curves, but they show more definite maxima in the range from pH 6.0 to pH 7.0. The broad plateau of the thermal enzyme curve may be limited by the “dibasic” nature of the substrate. However, enzyme-substrate experiments suggest that both enzyme and substrate are “dibasic” in nature, acting together to complicate the pH activity behavior. TABLE II Effect of Activator Molar ratio of MgCe to NaPsO
0.098 0.311 0.469 0.625 0.937
1.100
to Substrate
Ratios
on Activity Per cent hydrolysis 1.1 3.5 94.5 86.3 75.0
9.0
THERMAL
ENZYMES.
Enzyme-Substrate
VIII
44.7
Relations
The thermal PPase was similar to the ATPase reported earlier (6) with respect to ratios of activating ion to substrate. With constant initial amounts of either pyrophosphate or magnesium ion, the activity reached a maximum when the molar ratio of magnesium to sodium pyrophosphate was approximately 0.5. The rate of hydrolysis was markedly lower whenever this ratio was above or below t’he optimum value. Table II illustrates this. (Recent observations in our laboratory indicate that the degree of purification of the enzyme influences the magnesium ratio. The optimum ratio increases as the purity of the enzyme preparat,ions is increased.) Lohmann (11) showed that the rate of hydrolysis of sodium pyrophosphate by muscle or liver extracts was greatest when the rat’io of magnesium to sodium pyrophosphate was about 2.0. Bauer (12) and Kunitz (3) have both found that in the case of yeast PPase the optimal ratio of magnesium to pyrophosphate depends both on the pH and on the concentration of pyrophosphate. The optimum ratio for the t,hermal enzyme was apparently constant over a fairly wide pH range from about pH 5.5 to 9.5. Bloch-Frankenthal (13) has presented evidence that t,he true substrate for inorganic pyrophosphatase is the complex ion (MgP&)--. She found that for erythrocyte pyrophosphatase the optimum ratio of magnesium to substrate at pH 7.1 was approximately 3.8. Kunitz (3) was unable to evaluate the Michaelis constant, K, , for the yeast PPase. He used an excess of magnesium so that the concentration would be favorable even at high substrate concentrations, but did not obtain data consistent with the Linewearer-Burk (11) formulation for the estimation of K, . During the experiments with the t.hermal PPase the magnesiumsubstrat’e ratio was considered to be of more significance than t’he absolute magnesium concentration. The ratio was kept at a constant value of approximately 0.5 by preparing a stock solution of sodium pyrophosphate in magnesium chloride. The precipitation of ma.gnesium pyrophosphat,e was apparently prevented by the formation of a complex between magnesium and pyrophosphate. Van Wazer and Campanella (15) ha.ve presented good evidence that the alkaline-earth metals form strong chelate complexes with the various polyphosphates. In the process t,he weakly acidic hydrogens become more strongly dissociated. Thus
444
FIG.
CONNELL
2. Conventional represents molarity
MARSH
AND
WALTER
MILITZER
Lineweaver-Burk plot of substrate-activity of substrate and v micrograms P hydrolyzed/l5
data. S min.
the formation of the chelate complex may keep the phosphate in solution, competing with the formation of the magnesium salt. Incubation at 60°C. or higher may tend to break up the complex and accelerate precipitation. Digestion mixtures incubated at 60%. began to show some precipitation with pyrophosphate concentrations above 0.007 M. Due to the precipitation of the substrate at higher concentrations, a complete Michaelis curve could not be realized. However, as with the thermal ATPase (6), the values obtained were plotted, using the Lineweaver-Burk formulation. The (8)/u versus (8) plot (Fig. 2) gave a curve which was distinctly concave upward, instead of the expected straight line. Lineweaver and Burk have suggested that the assumption of only one substrate molecule in the ES complex may not be correct. That is, instead of E + S ---t ES -+ E + products
one may actually have E + NS --t ES,, -+ E f products
in which case the conventional plot of (S)/ZJ versus (S) gives a curve which is concave upward. If a straight line is to be obtained, plots of (S)*/v versus (S)% for various values of n must, be tried. The optimum magnesium-substratse ratio of 0.5 suggested that n in t’he case of the PPase might be 2. A plot of (S)2/v versus (S)z was made, giving the straight line of Fig. 3. The value of K, estimat,ed from the slope of this line was 5 X W3 M. Bailey (4) gave a value of 3.0 X 1W M for a highly purified yeast preparation, while Bloch-Frankent,hal (13) obtained a K, of 5.4 X W4 for erythrocyte pyrophosphat’ase. The calculated value of V,, for the thermal enzyme W,ZP 1160 pg. phosphorus/l5 min., which was a reasonable value. The optimum magnesium-substrate ratio of 0.5 and t)he indication t.hat two molecules of pyrophosphate are involved in the enzym+sl~l~strate romplex constitute fair evidence that t,he formation of a chelated metallosubstrat,e is necessary to t.he optimal catalytic activity of the thermal pyrophosphatase. The evidence is in rather close agreement with the metal1osubstrat.e theory of Najjar (16). The activating ion probably makes the pyrophosphate molecules more polar by chelate complex formation, so that they are in a configurat,ion a.ppropriate fol binding to the catalytic sit’es of t’he enzymes.
446
CONNELL
MARSH AND WALTER
MILITZER
Thernud Inactivation The heat-inactivation curves (Fig. 4) for the thermal PPase resemble curves for the general enzymatic decomposition of substrate by hydrolytic enzymes at high concentrations of substrate. The long period during which no inactivation is apparent suggests the presence of an inhibitor of the denaturation process. It may be that during the latent period the inhibitor is being removed or destroyed, after which the inactivation of the enzyme itself becomes apparent. None of the other enzymes of thermophile No. 2184 gave inactivation curves of this type. A transaminase preparation (17) gave curves which closely followed first-order kinetics throughout the process, as did the initial portion of curves for aldolase from the same source (18). Inactivation curves for crystalline yeast PPase (3) had the same general characteristics as those of the thermal enzyme. Possibly the observed kinetics of both enzymes are due to some factor inherent in the enzyme itself rather than to a separate protective entity.
FIQ. 4. Heat inactivation
of the thermal
inorganic
pyrophosphatase.
THERMAL
FIG.
5. Arrhenius
ENZYMES.
VIII
plot of thermal inactivation of inactivation.
data for ZO-min. period
Inactivation curves for the PPase, using a preparation considerably less pure than the one reported above, showed no greater heat stability, and the same latent period before denaturation (Fig. 5). Unless the prot’ective substance were very intimately associated with the enzyme purification such as was carried out should have altered the heat stability of the preparation. Free energy of activation, AF$, entropy of activation, A$, and heat of activation, AH’ for the process of inactivation were calculated by utilizing the equation from the theory of absolute reaction rates (19): k,
=
#T/h)
,As~/R
e-E/RT
where lz, = the first-order velocity constant K = the Boltzmann constant h = the Planck constant R = the universal gas constant T = absolute temperature. The inactivation process did not follotv exact first-order kinetics. However, rate constants were calculated upon assumed first-order be-
448
CONNELL
MAR8H
AND
TABLE Apparent Temp. “C.
Thermodynamic
75 70 65
18.021 8.650 4.310 1.911
(Kunitz) 60
0.0140
80
Twenty-Minute
Inactivation
Period
.931./m&
AH* cal./mole
29,700 28,780 31,190 30,000
34,606 34,626 34,723 34,250
13.9 16.8 10.3 12.6
0.14 0.16 0.10 0.12
8,276
51,929
71
0.45
AFf
x IO-6
MILITZER
III
for
Values
kr
sec.-
WALTER
sec.-l
A.9
TA.+/AHA
Cd./VdC~dCf.
havior. Rate constants were calculated using short intervals for the first 20- and 40-min. periods during which inactivation became apparent at each temperature. E for the 20min. period was 34,970 f 500 cal./mole. A plot similar to that of Fig. 5 gave as a value for the 40-min. period 44,237 f 500 cal./mole. The apparent thermodynamic values calculated for the 20-min. interval are given in Table III. Values calculated from Kunitz’ data are included for comparative purposes. His rate constants were also based upon assumed first-order behavior. Our AF$ and AHt: values are similar in magnitude to tabulated values for the denaturation of a variety of proteins. The AS’ values are considerably lower than ASf values for less heat-stable proteins. Stearn (20) gives as an average usual value for the ratio TASt/AHS of 0.36, whereas the average value for the thermal enzyme was about 0.13 for the 20-n&. inactivation period. The 40-min. values are shown in Table IV. Comparison with the 20min. values shows that while AF’ remained essentially constant, both AH’ and A$ increased rapidly. During 40 min. of inactivation TASf/AHf averaged about 0.35, close to the average literature values for protein denaturation. The AFf and AHf values are still within the usual range, but the average AS’ value of about 45.0 cal./mole/deg. is TABLE Apparent
Thermodynamic
Values
for
IV Forty-Minute
Temp.
k,
AF*
AH*
“C.
sec.-’
cal./mole
col./molr
27,904 28,652 28,600 28,269
44,142 42,920 43,898 44,423
80 75 70 65
9.75 3.37 1.19 5.76
x x x X
10-s 10-h 10-s 1OP
Inactivation As*
Period TAS’/AE
cal./mole/deg.
46.3 40.7 44.6 47.8
0.37 0.33 0.35 0.36
f
THERMAL
ENZYMES.
449
VIII
still significantly low, especially when compared to a value of 71.0 cal./mole/deg. for the yeast PPase. Stearn (20) has suggested the following equation for use in calculating the number of bonds broken during denaturation in processes where E is lower than 50,000-60,000 cal./mole: number
of
bonds
=
AHS.
-
20,000
4Qoo
fl
The value of 4000 Cal. is taken arbitrarily as the strength of weaker bonds broken, while the 20,000 cal. represents the energy involved in the initial hydrolytic breaking of a cystine bridge. For t’he thermophile PPase the equation yields 3-4 and 5-6 bonds for 20- and 40-min. inactivation intervals, respectively. For t’he yeast enzyme it gives 7-8 bonds. Thus it would appear that roughly twice as many bonds are broken in the yeast PPase as in the thermal PPase dur-
FIG. 6. Effect
of temperature Activit>is given
on catalytic as per cent
activity hydrolysis.
of the enzyme.
450
CONNELL
MARSH
AND
WALTER
MILITZER
ing comparable periods of inactivation. This suggests that perhaps there are inherent structural differences in the thermal enzyme molecules which may render them less vulnerable to heat denaturation. We fully realize the weakness of speculations of this nature from kinetic data. Perhaps we can be excused on the basis that no structural differences have yet been found to account for the great resistance to heat on the part of certain enzymes of which those from thermophilic bacteria are the leading exponents. Kinetics offers one approach which must be explored, along with others, in trying to find some clue toward explaining why the same enzyme from different sources can vary so widely in its resistance to inactivation by heat. Activation Energy Figure 6 illustrates the effect of temperature upon the catalytic activity of the PPase. Maximum activity represents complete hydrolysis of the substrate present. With optimal concentrations of substrate and magnesium the reaction went rapidly to completion even at 50°C. Activation energies were calculated from the data in Fig. 6 by means of the Arrhenius equation. The activation energy in the range 45-70°C. was 21,000 Cal., and 34,400 below 45°C. The definite break in the curve near 45°C. seems particularly significant since thermophile No. 2184 ceases to grow abundantly below this temperature. Kunitz (3) found a similar shift in the value for crystalline yeast PPase. fhKMARY
A highly active inorganic pyrophosphatare from a thermophilic bacterium has been partially purified and studied in some deiai:aiI. The enayme was dependent upon magnesium ions for activation and was activated to a lesser extent by cobalt. Neither calcium nor manganese was effective as an activating ion. The pH-activity curve was quite broad and exhibited a distinct plateau in the range from pH 5.5 to pH 9.5. The usual Lineweaver-Burk plot of (8)/v versus (S) did not give a straight line. Plotting (JS’)~/v versus (S)2 gave a straight line from which K, was calculated to be 5 X 10-8. Evidence is presented that the formation of a complex metallosubstrate is necessary for the hydrolysis of pyrophosphate by the enzyme. The energy of activation was found to be 21,000 Cal/mole in the range from 45 to 70°C. and 34,400 cal./mole in the range 30-45”C.
THERMAL
ENZYMES.
VIII
451
iIpparent thermodynamic values were calculated from heat-inactivation data. The most unusual of t,hese were t,he entropies of activatiolr, A#. For a 20-min. period of inactivation the mean AS’ value was 13.0 cal./mole/deg. After a 40-min. period of inactivation, the AS’ value \vas 45 cal./mole/deg. The low AS’ values suggest that there may be inherent structural differences in the t’hermal enzymes which render them less vulnerable to the denaturing effects of heat. REFERENCES
W., TUTTLE, L. C., AND GEORGI, C. E., ilrck. Biochem. and Biophys. 31, 416 (1951). MILITZER, W., AND TUTTLE, L. C., Arch. Biochem. and Biophys. 39,379 (1952). KUNITZ, M., J. Gen. Physiol. 33,423 (1952). BAILEY, K., AND WEBB, E. C., Biochem. J., 38,394 (1944). MILITZER, W. E., SONDEREQC+ER, T. B., TUTTLE, L. C., AND GEORGI, C. E., Arch. Biochem. 24, 75 (1949). MARSH, C. L., AND MILITZER, W. E., Arch. Biochem. and Biophys. 60, 433 (1956). FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 88,375 (1925). SWANSON, M. A., J. Biol. Chem. 191,577 (1951). ZELLER, E. A., Arch. Biochem. 28,138 (1950). KRISHNAN, P. S., Arch. Biochem. and Biophys. 37, 224 (1959). LOHMANN, K., Biochem. 2.262,137 (1933). BAUER, E., 2. physiol. Chem. 239,195 (1936). BLOCH-FRANKENTHAL, L., Biochem. J. 67,87 (1964). LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66,658 (1934). VAN WAZER, J. R., AND CAMPANELLA, D., J. Am. Chem. Sot. 72,655 (1950). NAJJAR, V. A., “A Symposium on Phosphorus Metabolism.” The Johns Hopkins University Press, Baltimore, Md., 1951. LINDSAY, H. L., Masters Thesis, University of Nebraska, 1952. BEINDORFF, A. B., Ph.D. Thesis, University of Nebraska, 1952. GLASSTONE, S., LAIDLER, K. J., AND EYRINO, II., “The Theory of Rate Processes.” McGraw-Hill, Inc., New York and London, 1941. STEARN, A. E., Advances in Enzymol. 9,37 (1949). LAIDLER, K. J., AND CASEY, E. S., J. Am. Chem. Sot. 72,2159 (1950). OUILLET, L., LAIDLER, K. J., AND MORALES, iif. F., Arch. Biochem. and Biophys. 39, 37 (1952).
1. MILITZER, 2.
3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. IO.
20. 21. 22.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
60,
439-451
(1956)
Thermal Enzymes. VIII. Properties of a Heat-Stable Inorganic Pyrophosphatasel Connell Marsh2 and Walter From the Department
of Chemistry,
University
Received February
Militzer
of Nebraska, Lincoln,
Nebraska
7, 1955
Soluble fractions from specific cell granules of a thermophilic bacterium, (NCA No. 2184), have been shown to contain apyrase and adenosinetriphosphatase (ATPase) (1, 2). During the course of experiments with the ATPase, a highly active inorganic pyrophosphatase (PPase) was found to be intimately associated with this enzyme. The PPase is activated by magnesium and, in general, resembles the inorganic PPase from yeast (3, 4) in its catalytic properties. The resistance of the thermophile enzyme, however, to denaturation by heat is in marked contrast to the heat lability of the yeast enzyme. The thermophile PPase is stable for almost an hour at 75°C. while the yeast PPase is rapidly inactivated at temperatures above 40°C. The kinetic and thermal data obtained by Kunitz for the crystalline yeast enzyme provided st,andards of comparison for the various properties of the PPase from our thermophile. This communication is devoted to a report on some of the properties of the thermal PPase, including metal-ion activation, enzyme-substrate behavior, effect of pH on activity, and thermal
and kinetic data. Values for AR”, AHt, A#, and E have been calculated from thermalinact.ivation data and, wherever possible, compared to values calculated from
Kunit.z’
data
(3).
1 Aided by a grant from the Division of Research Grants and Fellowships, U. S. Public Health Service, National Institutes of Health. 2 Present address: Department of Animal Pathology and Hygiene, College of .1griculture, University of Nebraska, Lincoln, Nebraska; 439
440
CONNELL
MARSH AND WALTER
MILITZER
EXPERIMENTAL
Preparation of PPase Growth and harvest of the thermophiles were conducted as described in a previous paper of ,this series (5). The preparation of granule suspensions, and the extraction and partial purification of PPase was essentially the same as reported earlier for the ATPase (1,6). For extraction of the PPase, the granule suspensions containing approximately 40 mg. protein/ml. were allowed to age in the refrigerator for about a week before fractionation. This procedure gave a better yield of enzyme. Similar to ATPase, the PPase activity was found in the precipitate from both the 0.35 and 0.5 saturated ammonium sulfate fractions following preliminary separations with acetone. The 0.35 fraction was less highly colored than the 0.5 fraction and usually contained less protein. The best PPase preparations gave Q,, values of from 15,000 to 25,060 and contained from 3 to 5 mg. protein/ml. The PPase was found to be inactivated by freezing and thawing but remained stable for months if kept at refrigerator temperatures without freezing.
Determination of Enzyme Activity Enzymatic activity was determined by measuring the amount of orthophosphorus liberated, using the calorimetric method of Fiske and SubbaRow (7). Standard PPase incubation mixtures contained 1.0 ml. of pH 7.5 borate buffer, 0.3 ml. of 0.032 M sodium pyrophosphate, 0.3 ml. of 0.015 M magnesium chloride (or magnesium sulfate), 0.1 ml. enzyme extract, and sufficient glass-distilled water to make a final volume of 1.8 ml. The mixtures were incubated at 60°C. for 15 min., after which the tubes were immersed in ice water, and 0.2 ml. of 5Oyo trichloroacetic acid was added to stop the reaction and to precipitate the protein. The precipitate was removed by filtration, and suitable aliquots were pipetted into 25-ml. volumetric flasks for the colorimetric determinations. Duplicate controls were included in each set of determinations. The enzyme in the controls was inactivated by immersing the tubes in boiling water for 5 min.
Procedure for Thermal Inactivation Studies One-tenth milliliter aliquots of the enzyme preparation were pipetted into thin-walled 18 X 150 mm. Pyrex tubes containing 1.0 ml. of 0.067 M borate buffer, pH 7.5, and 0.1 ml. of distilled water, giving a final protein concentration of 0.25 mg./ml. At zero time the tubes were immersed in a constant-temperature water bath, two tubes being reserved for determination of the activity of the unheated enzyme. After appropriate time intervals the tubes were withdrawn and cooled in an ice bath. To each of the tubes were added 0.3 ml. of 0.032 M sodium pyrophosphate and 0.3 ml. of 0.02 M magnesium sulfate, after which they were incubated for 15 min. at 60°C. The difference between the zero-time activity and the activity after heating was used as the measure of inactivation of the enzyme.
THERMAL
ENZYMES. TABLE
E$ect
of Various
Ions
upon
VIII
441
I
Activity
of Pyrophosphntase
Incubation mixtures contained 1.0 ml. borate buffer, pH 7.5; 0.3 ml. sodium pyrophosphate, 0.032 M; 0.1 ml. enzyme solution; 0.1 ml. distilled water; and 0.3 ml. activating ion solution. Activit,ies are expressed as micrograms P released in 15 min. at 60°C. Activating
hIgSO*
(0.02
ion
.ktivity
M)
595
CoClz (0.02 M) hfnSOl (0.02 M) CaC12 (0.02 M] RESULTS
110 0 0 AND DISCUSSION
General The PPase showed a positive requirement for magnesium as an activating ion. There was no measurable activity where magnesium was omitted from the incubation mixtures. There was some activation by cobalt, but none by manganese or calcium. Table I shows the relative efficiency of these ions as activators. Crystalline PPase isolated by Kunitz from yeast (3) was activated best by magnesium and was also activated by cobalt and manganese, while calcium was inhibitory. Specificity Most of the PPase preparations also contained ATPase, and some would hydrolyze sodium tripolyphosphate slightly. There was no h;vdrolytic action on sodium trimetaphosphate and no phosphomonoesterase activity. Swanson (S), Zeller (9), and Krishnan (10) have suggested that in view of the marked similarities of ATPases and PPases, which are activated by magnesium, these enzymes may possibly be one and thr same ent,ity, at least in some instances. Krishnan obtained essentially constant PPase-apyrase activity ratios at various stages of purification of extracts from Penicillium chrysogenum. He concluded that one enzyme was responsible for t’he hydrolysis of both adenosine triphosphatc (ATP) and pyrophosphate. Similar studies with the thermophile e11zymes showed no such correlation of activities. Some fractions VOW tained ATPase and no PPase, and vice versa, while other fractions caontainedboth activities. Furt’hermore, preparations whirh initially con-
442
CONNELL
MARSH
AND
WALTER
FIG. 1. Effect of pH on enzyme activity;
MILITZER
Verona1 buffer, 0.10 M.
tained both ATPase and PPase rapidly lost their PPase activity upon repeated freezing and thawing, or during storage in the frozen state. The thermophile enzymes thus appear to be distinctly separate entities. The pH-activity curve for the thermal enzyme (Fig. 1) is broad and flat, somewhat like the curves obtained by Krishnan (10). Kunitz obtained rather broad pH curves, but they show more definite maxima in the range from pH 6.0 to pH 7.0. The broad plateau of the thermal enzyme curve may be limited by the “dibasic” nature of the substrate. However, enzyme-substrate experiments suggest that both enzyme and substrate are “dibasic” in nature, acting together to complicate the pH activity behavior. TABLE II Effect of Activator Molar ratio of MgCe to NaPsO
0.098 0.311 0.469 0.625 0.937
1.100
to Substrate
Ratios
on Activity Per cent hydrolysis 1.1 3.5 94.5 86.3 75.0
9.0
THERMAL
ENZYMES.
Enzyme-Substrate
VIII
44.7
Relations
The thermal PPase was similar to the ATPase reported earlier (6) with respect to ratios of activating ion to substrate. With constant initial amounts of either pyrophosphate or magnesium ion, the activity reached a maximum when the molar ratio of magnesium to sodium pyrophosphate was approximately 0.5. The rate of hydrolysis was markedly lower whenever this ratio was above or below t’he optimum value. Table II illustrates this. (Recent observations in our laboratory indicate that the degree of purification of the enzyme influences the magnesium ratio. The optimum ratio increases as the purity of the enzyme preparat,ions is increased.) Lohmann (11) showed that the rate of hydrolysis of sodium pyrophosphate by muscle or liver extracts was greatest when the rat’io of magnesium to sodium pyrophosphate was about 2.0. Bauer (12) and Kunitz (3) have both found that in the case of yeast PPase the optimal ratio of magnesium to pyrophosphate depends both on the pH and on the concentration of pyrophosphate. The optimum ratio for the t,hermal enzyme was apparently constant over a fairly wide pH range from about pH 5.5 to 9.5. Bloch-Frankenthal (13) has presented evidence that t,he true substrate for inorganic pyrophosphatase is the complex ion (MgP&)--. She found that for erythrocyte pyrophosphatase the optimum ratio of magnesium to substrate at pH 7.1 was approximately 3.8. Kunitz (3) was unable to evaluate the Michaelis constant, K, , for the yeast PPase. He used an excess of magnesium so that the concentration would be favorable even at high substrate concentrations, but did not obtain data consistent with the Linewearer-Burk (11) formulation for the estimation of K, . During the experiments with the t.hermal PPase the magnesiumsubstrat’e ratio was considered to be of more significance than t’he absolute magnesium concentration. The ratio was kept at a constant value of approximately 0.5 by preparing a stock solution of sodium pyrophosphate in magnesium chloride. The precipitation of ma.gnesium pyrophosphat,e was apparently prevented by the formation of a complex between magnesium and pyrophosphate. Van Wazer and Campanella (15) ha.ve presented good evidence that the alkaline-earth metals form strong chelate complexes with the various polyphosphates. In the process t,he weakly acidic hydrogens become more strongly dissociated. Thus
444
FIG.
CONNELL
2. Conventional represents molarity
MARSH
AND
WALTER
MILITZER
Lineweaver-Burk plot of substrate-activity of substrate and v micrograms P hydrolyzed/l5
data. S min.
the formation of the chelate complex may keep the phosphate in solution, competing with the formation of the magnesium salt. Incubation at 60°C. or higher may tend to break up the complex and accelerate precipitation. Digestion mixtures incubated at 60%. began to show some precipitation with pyrophosphate concentrations above 0.007 M. Due to the precipitation of the substrate at higher concentrations, a complete Michaelis curve could not be realized. However, as with the thermal ATPase (6), the values obtained were plotted, using the Lineweaver-Burk formulation. The (8)/u versus (8) plot (Fig. 2) gave a curve which was distinctly concave upward, instead of the expected straight line. Lineweaver and Burk have suggested that the assumption of only one substrate molecule in the ES complex may not be correct. That is, instead of E + S ---t ES -+ E + products
one may actually have E + NS --t ES,, -+ E f products
in which case the conventional plot of (S)/ZJ versus (S) gives a curve which is concave upward. If a straight line is to be obtained, plots of (S)*/v versus (S)% for various values of n must, be tried. The optimum magnesium-substratse ratio of 0.5 suggested that n in t’he case of the PPase might be 2. A plot of (S)2/v versus (S)z was made, giving the straight line of Fig. 3. The value of K, estimat,ed from the slope of this line was 5 X W3 M. Bailey (4) gave a value of 3.0 X 1W M for a highly purified yeast preparation, while Bloch-Frankent,hal (13) obtained a K, of 5.4 X W4 for erythrocyte pyrophosphat’ase. The calculated value of V,, for the thermal enzyme W,ZP 1160 pg. phosphorus/l5 min., which was a reasonable value. The optimum magnesium-substrate ratio of 0.5 and t)he indication t.hat two molecules of pyrophosphate are involved in the enzym+sl~l~strate romplex constitute fair evidence that t,he formation of a chelated metallosubstrat,e is necessary to t.he optimal catalytic activity of the thermal pyrophosphatase. The evidence is in rather close agreement with the metal1osubstrat.e theory of Najjar (16). The activating ion probably makes the pyrophosphate molecules more polar by chelate complex formation, so that they are in a configurat,ion a.ppropriate fol binding to the catalytic sit’es of t’he enzymes.
446
CONNELL
MARSH AND WALTER
MILITZER
Thernud Inactivation The heat-inactivation curves (Fig. 4) for the thermal PPase resemble curves for the general enzymatic decomposition of substrate by hydrolytic enzymes at high concentrations of substrate. The long period during which no inactivation is apparent suggests the presence of an inhibitor of the denaturation process. It may be that during the latent period the inhibitor is being removed or destroyed, after which the inactivation of the enzyme itself becomes apparent. None of the other enzymes of thermophile No. 2184 gave inactivation curves of this type. A transaminase preparation (17) gave curves which closely followed first-order kinetics throughout the process, as did the initial portion of curves for aldolase from the same source (18). Inactivation curves for crystalline yeast PPase (3) had the same general characteristics as those of the thermal enzyme. Possibly the observed kinetics of both enzymes are due to some factor inherent in the enzyme itself rather than to a separate protective entity.
FIQ. 4. Heat inactivation
of the thermal
inorganic
pyrophosphatase.
THERMAL
FIG.
5. Arrhenius
ENZYMES.
VIII
plot of thermal inactivation of inactivation.
data for ZO-min. period
Inactivation curves for the PPase, using a preparation considerably less pure than the one reported above, showed no greater heat stability, and the same latent period before denaturation (Fig. 5). Unless the prot’ective substance were very intimately associated with the enzyme purification such as was carried out should have altered the heat stability of the preparation. Free energy of activation, AF$, entropy of activation, A$, and heat of activation, AH’ for the process of inactivation were calculated by utilizing the equation from the theory of absolute reaction rates (19): k,
=
#T/h)
,As~/R
e-E/RT
where lz, = the first-order velocity constant K = the Boltzmann constant h = the Planck constant R = the universal gas constant T = absolute temperature. The inactivation process did not follotv exact first-order kinetics. However, rate constants were calculated upon assumed first-order be-
448
CONNELL
MAR8H
AND
TABLE Apparent Temp. “C.
Thermodynamic
75 70 65
18.021 8.650 4.310 1.911
(Kunitz) 60
0.0140
80
Twenty-Minute
Inactivation
Period
.931./m&
AH* cal./mole
29,700 28,780 31,190 30,000
34,606 34,626 34,723 34,250
13.9 16.8 10.3 12.6
0.14 0.16 0.10 0.12
8,276
51,929
71
0.45
AFf
x IO-6
MILITZER
III
for
Values
kr
sec.-
WALTER
sec.-l
A.9
TA.+/AHA
Cd./VdC~dCf.
havior. Rate constants were calculated using short intervals for the first 20- and 40-min. periods during which inactivation became apparent at each temperature. E for the 20min. period was 34,970 f 500 cal./mole. A plot similar to that of Fig. 5 gave as a value for the 40-min. period 44,237 f 500 cal./mole. The apparent thermodynamic values calculated for the 20-min. interval are given in Table III. Values calculated from Kunitz’ data are included for comparative purposes. His rate constants were also based upon assumed first-order behavior. Our AF$ and AHt: values are similar in magnitude to tabulated values for the denaturation of a variety of proteins. The AS’ values are considerably lower than ASf values for less heat-stable proteins. Stearn (20) gives as an average usual value for the ratio TASt/AHS of 0.36, whereas the average value for the thermal enzyme was about 0.13 for the 20-n&. inactivation period. The 40-min. values are shown in Table IV. Comparison with the 20min. values shows that while AF’ remained essentially constant, both AH’ and A$ increased rapidly. During 40 min. of inactivation TASf/AHf averaged about 0.35, close to the average literature values for protein denaturation. The AFf and AHf values are still within the usual range, but the average AS’ value of about 45.0 cal./mole/deg. is TABLE Apparent
Thermodynamic
Values
for
IV Forty-Minute
Temp.
k,
AF*
AH*
“C.
sec.-’
cal./mole
col./molr
27,904 28,652 28,600 28,269
44,142 42,920 43,898 44,423
80 75 70 65
9.75 3.37 1.19 5.76
x x x X
10-s 10-h 10-s 1OP
Inactivation As*
Period TAS’/AE
cal./mole/deg.
46.3 40.7 44.6 47.8
0.37 0.33 0.35 0.36
f
THERMAL
ENZYMES.
449
VIII
still significantly low, especially when compared to a value of 71.0 cal./mole/deg. for the yeast PPase. Stearn (20) has suggested the following equation for use in calculating the number of bonds broken during denaturation in processes where E is lower than 50,000-60,000 cal./mole: number
of
bonds
=
AHS.
-
20,000
4Qoo
fl
The value of 4000 Cal. is taken arbitrarily as the strength of weaker bonds broken, while the 20,000 cal. represents the energy involved in the initial hydrolytic breaking of a cystine bridge. For t’he thermophile PPase the equation yields 3-4 and 5-6 bonds for 20- and 40-min. inactivation intervals, respectively. For t’he yeast enzyme it gives 7-8 bonds. Thus it would appear that roughly twice as many bonds are broken in the yeast PPase as in the thermal PPase dur-
FIG. 6. Effect
of temperature Activit>is given
on catalytic as per cent
activity hydrolysis.
of the enzyme.
450
CONNELL
MARSH
AND
WALTER
MILITZER
ing comparable periods of inactivation. This suggests that perhaps there are inherent structural differences in the thermal enzyme molecules which may render them less vulnerable to heat denaturation. We fully realize the weakness of speculations of this nature from kinetic data. Perhaps we can be excused on the basis that no structural differences have yet been found to account for the great resistance to heat on the part of certain enzymes of which those from thermophilic bacteria are the leading exponents. Kinetics offers one approach which must be explored, along with others, in trying to find some clue toward explaining why the same enzyme from different sources can vary so widely in its resistance to inactivation by heat. Activation Energy Figure 6 illustrates the effect of temperature upon the catalytic activity of the PPase. Maximum activity represents complete hydrolysis of the substrate present. With optimal concentrations of substrate and magnesium the reaction went rapidly to completion even at 50°C. Activation energies were calculated from the data in Fig. 6 by means of the Arrhenius equation. The activation energy in the range 45-70°C. was 21,000 Cal., and 34,400 below 45°C. The definite break in the curve near 45°C. seems particularly significant since thermophile No. 2184 ceases to grow abundantly below this temperature. Kunitz (3) found a similar shift in the value for crystalline yeast PPase. fhKMARY
A highly active inorganic pyrophosphatare from a thermophilic bacterium has been partially purified and studied in some deiai:aiI. The enayme was dependent upon magnesium ions for activation and was activated to a lesser extent by cobalt. Neither calcium nor manganese was effective as an activating ion. The pH-activity curve was quite broad and exhibited a distinct plateau in the range from pH 5.5 to pH 9.5. The usual Lineweaver-Burk plot of (8)/v versus (S) did not give a straight line. Plotting (JS’)~/v versus (S)2 gave a straight line from which K, was calculated to be 5 X 10-8. Evidence is presented that the formation of a complex metallosubstrate is necessary for the hydrolysis of pyrophosphate by the enzyme. The energy of activation was found to be 21,000 Cal/mole in the range from 45 to 70°C. and 34,400 cal./mole in the range 30-45”C.
THERMAL
ENZYMES.
VIII
451
iIpparent thermodynamic values were calculated from heat-inactivation data. The most unusual of t,hese were t,he entropies of activatiolr, A#. For a 20-min. period of inactivation the mean AS’ value was 13.0 cal./mole/deg. After a 40-min. period of inactivation, the AS’ value \vas 45 cal./mole/deg. The low AS’ values suggest that there may be inherent structural differences in the t’hermal enzymes which render them less vulnerable to the denaturing effects of heat. REFERENCES
W., TUTTLE, L. C., AND GEORGI, C. E., ilrck. Biochem. and Biophys. 31, 416 (1951). MILITZER, W., AND TUTTLE, L. C., Arch. Biochem. and Biophys. 39,379 (1952). KUNITZ, M., J. Gen. Physiol. 33,423 (1952). BAILEY, K., AND WEBB, E. C., Biochem. J., 38,394 (1944). MILITZER, W. E., SONDEREQC+ER, T. B., TUTTLE, L. C., AND GEORGI, C. E., Arch. Biochem. 24, 75 (1949). MARSH, C. L., AND MILITZER, W. E., Arch. Biochem. and Biophys. 60, 433 (1956). FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 88,375 (1925). SWANSON, M. A., J. Biol. Chem. 191,577 (1951). ZELLER, E. A., Arch. Biochem. 28,138 (1950). KRISHNAN, P. S., Arch. Biochem. and Biophys. 37, 224 (1959). LOHMANN, K., Biochem. 2.262,137 (1933). BAUER, E., 2. physiol. Chem. 239,195 (1936). BLOCH-FRANKENTHAL, L., Biochem. J. 67,87 (1964). LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66,658 (1934). VAN WAZER, J. R., AND CAMPANELLA, D., J. Am. Chem. Sot. 72,655 (1950). NAJJAR, V. A., “A Symposium on Phosphorus Metabolism.” The Johns Hopkins University Press, Baltimore, Md., 1951. LINDSAY, H. L., Masters Thesis, University of Nebraska, 1952. BEINDORFF, A. B., Ph.D. Thesis, University of Nebraska, 1952. GLASSTONE, S., LAIDLER, K. J., AND EYRINO, II., “The Theory of Rate Processes.” McGraw-Hill, Inc., New York and London, 1941. STEARN, A. E., Advances in Enzymol. 9,37 (1949). LAIDLER, K. J., AND CASEY, E. S., J. Am. Chem. Sot. 72,2159 (1950). OUILLET, L., LAIDLER, K. J., AND MORALES, iif. F., Arch. Biochem. and Biophys. 39, 37 (1952).
1. MILITZER, 2.
3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. IO.
20. 21. 22.