2212
Biochemistry 1988, 27, 2212-2217
Involvement of Arginine Residues in the Activation of Calmodulin-Dependent 3',5'-Cyclic-Nucleotide Phosphodiesterase Narasimha Nibhanupudy, Frederick Jones, and Allen R . Rhoads* Department of Biochemistry, Howard University College of Medicine, Washington, D.C. 20059 Received December 30, 1986; Revised Manuscript Received November 19, 1987
ABSTRACT: Pretreatment of an affinity-purified, brain calmodulin (CaM)-dependent phosphodiesterase (EC
3.1.4.17) with p-hydroxyphenylglyoxal (pHPG), a specific arginine-modifying reagent, resulted in a time-dependent loss in CaM-stimulated hydrolysis of cyclic A M P and cyclic G M P with no change in basal, CaM-independent activity. The loss in CaM-stimulated activity was preceded by a transient increase in CaM-dependent activity. Phenylglyoxal was 10-fold more effective than pHPG in promoting the loss of CaM-stimulated activity with a second-order rate constant of 13.3 M-' min-'. Other arginine-modifying reagents, 1,2-~yclohexanedioneand 2,3-butanedione, were not effective. The pHPG-modified enzyme was activated by 100 FM lysophosphatidylcholine to levels comparable to CaM-stimulated activity. The arginyl-modified enzyme was also activated by chymotrypsin and trypsin but not to the extent of the untreated enzyme stimulated with CaM. The presence of C a M during chemical modification with pHPG protected the enzyme from inactivation. Both the extent of activation and the amount of C a M necessary for 50% maximal activation were affected by pHPG treatment of the enzyme. The approximate number of modified arginines estimated by [7-'4C]phenylglyoxal incorporation and amino acid analysis after complete inactivation of C a M stimulation was seven residues per catalytic subunit assuming enzyme homogeneity. The Stokes radius and sedimentation coefficient of the enzyme were unchanged by the modification. These results suggest that arginine residues are critical for functional interaction between phosphodiesterase and C a M and that controlled modification can selectively alter CaM-stimulated enzyme activity.
T e binding of calcium to calmodulin (CaM)' results in conformational changes that permit specific association with discrete regions of target enzymes and proteins (Manalan & Nee, 1984). The calcium-induced structural changes in CaM facilitate binding of hydrophobic probes (LaPorte et al., 1980) and specific hydrophobic, cationic drugs (Weiss et al., 1982). In addition, CaM binds a number of naturally occurring peptides and toxins in a calcium-dependent manner (Malencik & Anderson, 1983; Cox et al., 1985), and studies of the binding of peptides suggest that both hydrophobic and electrostatic components stabilize the interaction (O'Neil & DeGrado, 1985). Although the physiological significance of the interaction between the bioactive polypeptides and CaM is not known, the structural features of these peptides are consistent with the presence of basic amino acid residues, a low frequency of acidic residues, and adjacent hydrophobic sequences. These structural characteristics are also present in isolated CaMbinding peptides and homologous regions identified in CaMregulated enzymes (Blumenthal et al., 1985; Hanley et al., 1987) and in model peptides that bind to CaM with high affinity. A basic amphipathic helix on the target protein or peptide may be necessary for interaction with CaM (Cox et al., 1985; McDowell et al., 1985); however, studies of the interaction of troponin I and mastoparan with CaM or troponin C indicate that although a-helical structure may not be a prerequisite for binding, basic residues are important (Cachia, 1986). The specific binding regions on CaM-dependent target enzymes may recognize different structural elements of calcium-activated CaM (Newton et al., 1984), leading to interactive I Abbreviations: pHPG, p-hydroxyphenylglyoxal: PG, phenylglyoxal; CaM, calmodulin; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, ethylene glycol bis(@aminoethyl ether)-N,N,N',"-tetraacetic acid: EDTA, ethylenediaminetetraacetic acid: Tris, tris(hydroxymethy1)aminomethane.
0006-2960/88/0427-2212$01.50/0
diversity in the binding domain for CaM. The high-affinity binding of CaM to 3',5'-cyclic-nucleotide phosphodiesterase involves the association of two molecules of CaM with a dimer of catalytic subunits (LaPorte et al., 1979; Klee et al., 1979; Sharma et al., 1980). To investigate whether CaM-dependent phosphodiesterase possesses cationic residues essential for a CaM-binding domain, we examined the effect of the modification of arginine residues on CaM activation and on several physical properties of the CaM-dependent phosphodiesterase purified from brain cortex. MATERIALS AND METHODS pHPG was purchased from Pierce Chemical Co., Rockford, IL. Ethyl acetimidate was from Aldrich Chemical Co., Milwaukee, WI. Phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, pyridoxal phosphate, a-chymotrypsin (crystallized 3 times), and crystallized trypsin were purchased from Sigma Chemical Co., St. Louis, MO. Pronase was from Calbiochem, San Diego, CA. [ ''C]Phenylglyoxal (25 mCi/ mmol) was obtained from Amersham Corp., Arlington Heights, IL. CaM-Dependent Phosphodiesterase and CaM. CaM-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain through CaM affinity chromatography by slight modification of the procedure of Sharma et al. (1983). The final enzyme preparation was activated 7-lo-fold and had a specific activity of 10-20 units/mg of protein. The purified enzyme gave two major bands on denaturing polyacrylamide gel electrophoresis as described previously (Sharma et al., 1984). The isozyme subunit composition was estimated by densitometric analysis of gels stained with Coomassie Blue to be 55% and 45% for the M I 63 000 and 60 000 subunits, respectively. contaminating protein bands were also present at M I 45 000 and 36 000. The purified enzyme was further characterized by a molecular weight of 128 000 by gel filtration and a sedimentation coefficient of 5.5 S as determined by 0 1988 American Chemical Society
VOL. 27, NO. 6, 1988
ARGININE MODIFICATION OF PHOSPHODIESTERASE
sucrose density gradient centrifugation. The enzyme was extensively dialyzed against 10 mM HEPES buffer, pH 8.0, and 25% (v/v) glycerol and stored at -20 OC without loss in activity or CaM activation. CaM was purified from bovine brain cortex as described by Kakuichi et al. (1981) with modifications (Rhoads et al., 1985). The measurement of phosphodiesterase activity was determined colorimetrically by the release of phosphorus (Butcher & Sutherland, 1962). The final volume of the assay mixture was 500 pL and contained 40 mM Tris-HC1, pH 7.5, 5 mM MgC12, 2 mM cyclic AMP or cyclic GMP, and either 500 pM calcium with 1.0 pg of CaM (CaM-stimulated activity) or 1 mM EGTA (basal activity). Assay time was usually 20 min at 30 O C , and reactions were terminated by heating for 3 min at 100 OC followed by stoichiometric conversion of 5'-AMP to adenosine and inorganic phosphorus by Crotalus atrox venom (75 pg of protein/assay). All reagents and chemicals used in these experiments were tested at their final diluted concentrations in enzyme reaction mixtures for interference with the stoichiometric conversion of 200-500 nmol of 5'-AMP to phosphorus by venom 5'-nucleotidase. Protein was measured by the procedure of Bradford ( 1 976). Chemical Modification of Phosphodiesterase and CaM. The CaM-dependent phosphodiesterasewas preincubated with glyoxal derivatives in the dark at 30 "C in the presence of 80 mM sodium bicarbonate at pH 8.0 (Cheung & Fonda, 1979). The control and treated enzymes were incubated at 30-70 pg/mL in the presence of 1 mM EDTA and 3-6% (v/v) glycerol for the indicated time periods. Enzyme aliquots of control and treated mixtures were diluted 10-25-fold into the phosphodiesterase assay mixture. In [I4C]phenylglyoxal labeling experiments, the enzyme was incubated with 5 mM radiolabeled reagent with a final specific activity of 25 mCi/mmol. Reactions were terminated by a 50-fold dilution into 10 mM Tris-HC1, pH 7.5, and 10 mM arginine. Samples were dialyzed against several changes of 10 mM Tris-HC1, pH 7.5, buffer containing 100 mM NaCl over a 48-h period. Basal and control activities were measured after 10-20 min of incubation with 2 mM cyclic AMP. Preincubation mixtures were assayed in duplicate at each time point. Addition of excess arginine to quench the modification reaction did not reverse the inactivation process. Modification with 2,3-butanedione and 1,2-cyclohexanedioneemployed 50 mM borate, pH 8.0, and 100 mM borate, pH 9.0, in place of bicarbonate buffer, respectively. Enzyme concentration was 53.3 pg/mL, and other conditions were as described above. For amino acid analysis, the enzyme (250 pg) was reacted with 5 mM PG for 20 min under the above reaction conditions, and the mixtures were treated with sodium borohydride in a 10-fold molar excess of PG to terminate the reaction by reduction to the glycol. Treated and untreated control samples were extensively dialyzed against 0.1 M N a H C 0 3 buffer at pH 8.0 and deionized water before lyophilization and analysis. Amino acid analyses were performed on a Dionex amino acid analyzer equipped with a Model 120B Beckman-Spinco analyzer. For modification by pyridoxal phosphate, a 150-pL aliquot of enzyme was placed on a 0.6 X 7.0 cm column of Sephadex G-25 equilibrated with 40 mM HEPES, pH 7.8. The enzyme eluted from the column was pooled and preincubated at 26.2 pg of protein/mL with 2 mM pyridoxal phosphate in 40 mM HEPES, pH 7.8, and 0.1 mM EDTA at 30 OC. Samples were taken after different times and exposed to 13.9 mM NaBH, for 15 s and adjusted to 155 mM Tris-HC1, pH 7.5, and 1.5 mg/mL bovine serum albumin. These mixtures were placed
2213
.
1.0
E
a
2.
* *
:
1.0
-
I 0
' 1 0
2 0
INCUBATION
3 0
\5 0
T I M E , min.
Inactivation of CaM-deficient phosphodiesterase by pHPG. Enzyme (35.5 pg/mL) was incubated with or without 40 mM pHPG in 3.1% (v/v) glycerol at 30 OC. At different times, a 50-pL aliquot was removed for the determination of phosphodiesterase activity in the presence of 2 pg/mL CaM and untreated (0)or pHPG-treated enzyme ( 0 ) . The basal activity (m) of the treated enzyme was measured in the presence of 1.O mM EGTA. Enzyme activity was measured at 2 mM cyclic AMP over a 20-min period as described under Materials and Methods. FIGURE1:
on ice for 15 min prior to assay. Modification of the enzyme (53.3 pg/mL) by 20 mM ethyl acetimidate was performed in 50 mM borate, pH 8.5, and 1 mM EDTA at 30 OC. CaM (100 pg/mL) was treated with 40 mM pHPG in the presence of 80 mM bicarbonate buffer, pH 8.0, and 1 mM EDTA. After incubation for 25 min, the solution was diluted 10-fold, and aliquots ranging from 0 to 1.0 pg were added to enzyme assay mixtures to determine the extent of activation of CaM-deficient phosphodiesterase. In CaM protection experiments, enzyme (68.2 pg/mL) was first incubated for a period of 5 min at 30 OC with CaM (0-0.654 pg of CaM/pg of enzyme) and 250 pM calcium chloride. pHPG was then added to a final concentration of 40 mM and incubation continued for 40 min. All other conditions were as indicated for pHPG modification. In proteolysis experiments, the enzyme was pretreated with 40 mM pHPG at 227 pg/mL enzyme for 35 min and placed on a 0.6 X 8.0 cm Sephadex G-25 column equilibrated with 10 mM HEPES, pH 7.5, and 400-pL fractions were collected. Enzyme protein was pooled and incubated at 16.6 pg/mL with 0.55 pg/mL trypsin, a-chymotrypsin, or Pronase in 50 mM Tris-HC1, pH 8.0, 200 mM NaC1, and 5 mM MgCl,. After treatment for 10 min at 30 OC, aliquots were placed in assay tubes containing 50 pg of soybean trypsin inhibitor, and phosphodiesterase activity was measured.
RESULTS Treatment of CaM-deficient phosphodiesterase with 40 mM pHPG (Figure 1) resulted in a near-complete, time-dependent loss in CaM-stimulated activity without affecting basal activity. In the absence of pHPG, no loss in CaM-stimulated activity was observed. The inactivation of CaM-stimulated activity was preceded by a transient increase of up to 150% in the CaM-stimulated activity (Figure 2). When cyclic GMP was used as the substrate, time-dependent inactivation was similar to that shown for cyclic AMP as substrate, but the transient activation phase was diminished. Variation in the concentration of pHPG in the preincubation mixture from 0 to 60 mM resulted in a progressive increase in the level of transient activation and the rate of inactivation
2214
B IO C H E M I S T R Y
NIBHANUPUDY ET AL.
1
1 0
0
2 0
1 0
INCUBATION
TIME,
4 0
min
I
I 7
8
-log
6
6
(CaM)
FIGURE 3: Activation of pHPG-treated and untreated brain phosphodiesterase by CaM. The enzyme (56.8 Mg/mL) was pretreated in the presence ( 0 )or absence (0) of 40 mM pHPG for 35 min in 3.2% (v/v) glycerol at 30 OC under standard conditions. A 30-pL aliquot was then assayed for total phosphodiesteraseactivity at different CaM concentrations.
- 0.9
-1.1 v)
P 0
x
P
0
-1.3
-1.5
I
I
- 2.0
I
-1.5
I
-1.0
log ( p H P G ) , M Inactivation of CaM-stimulated brain phosphodiesterase activity as a function of pHPG concentration. Enzyme (35.5 pg/mL) was incubated at 30 "C with pHPG (0-60 mM) and 3.1% (v/v) glycerol under standard conditions. At the indicated times, 30-pL aliquots were removed and assayed as described under Materials and Methods. (A) Plot of CaM-stimulated activity (activated-basal) of treated enzyme expressed as percent of untreated control versus treatment time. (B) Plot of the observed rate constant for the inactivation phase of (A) with respect to the concentration of pHPG. The second-order rate constant and reaction dumber were determined from linear regression of this secondary plot. FIGURE 2:
(Figure 2A). The activation and inactivation phases were dependent upon the concentration of pHPG. A plot of In (% control X versus time gave pseudo-first-order kinetics for the inactivation phase (data not shown). A plot of the logarithm of the observed rate constants for each concentration of pHPG yielded a slope (reaction number) of 0.83 (Levy et al., 1963) and a second-order rate constant of 1.31 M-' min-* (Figure 2B). The slope and linearity of the plot indicate an apparent first-order inactivation of a single class of pHPG-
reactive sites. In similar studies, treatment of phosphodiesterase with the parent compound phenylglyoxal (PG), which also reacts specifically with the guanidyl group of arginine under mild conditions (Takahashi, 1977), indicated that this compound was approximately 10-fold more potent than pHPG in causing a loss in CaM-stimulated activity. A plot of the observed rate constant against PG concentration gave a reaction number of 1.01 and a second-order rate constant of 13.3 M-' min-'. An activation phase was observed, but at low concentrations (55 mM) of PG where the rate of inactivation apparently did not mask the activation phase. The time and concentration dependences of the inactivation were consistent with chemical modification of CaM-deficient phosphodiesterase. Treatment of CaM with pHPG for 25 min (not shown) did not affect the saturation isotherm for CaM activation of the enzyme. The pHPG-treated CaM was able to stimulate the activity of phosphodiesterase as effectively as untreated CaM. The level of activation and the concentration of CaM necessary for 50% activation were dramatically affected by treatment of the enzyme with 40 mM pHPG for 30 min (Figure 3). Preliminary experiments indicated that the rate of inactivation of CaM-stimulated activity by pHPG was the same regardless of the CAM concentration (0.1-10 pM) used to activate the treated enzyme, supporting the data in Figure 3 and suggesting that major changes in the ability of the enzyme to interact with CaM occur upon treatment with pHPG. Lysophosphatidylcholine was reported to cause a marked, saturable stimulation of phosphodiesterase activity comparable to activation of the enzyme by CaM (Pichard & Cheung, 1977). When the effects of CaM and lysophosphatidylcholine on the pHPG-treated enzyme were compared (Figure 4), no substantial change in the activation of the enzyme by lysophosphatidylcholine was seen throughout the course of pHPG treatment. CaM-dependent phosphodiesterase can also be activated in an irreversible manner by treatment with several proteases (Kincaid et al., 1985). Exposure of the pHPGtreated enzyme to protease action (Table I) led to only partial activation (57-67% of total activity) compared to protease treatment of the unmodified enzyme. For the untreated enzyme, protease activation was comparable to activation by CaM, alone.
ARGININE MODIFICATION OF PHOSPHODIESTERASE
0
1 0
2 0
INCUBATION
3 0 TIME, min
4 0
FIGURE 4: Effect of pHPG treatment on activation of phosphodiesterase by CaM or lysophosphatidylcholine. Enzyme (56.8 pg/mL) was incubated with 40 mM pHPG and 3.2% (v/v) glycerol under described conditions for different time periods. A 30-pL aliquot was assayed in the presence of 1.0 pg of CaM ( 0 ) or 100 pM lysophosphatidylcholine (0). The extent of activation of the untreated enzyme by these agents ranged from 6- to 7-fold.
VOL. 27, NO. 6, 1988
2215
Table I: Effect of Protease Treatment on pHPG-Modified Phosphodiesterase and Unmodified Enzyme activity (pmol min-' mL-I) treatment basal totalb modified enzyme" none 0.414 0.873 trypsin 1.24 1.16 chymotrypsin 0.752 0.967 Pronase 0.855 0.979 unmodified enzyme none 0.393 1.71 trypsin 1.83 1.65 chymotrypsin 1.16 1.69 Pronase 1.28 1.71 'Enzyme was pretreated with 40 mM pHPG for 35 min as described under Materials and Methods and compared with control enzyme incubated under identical conditions without pHPG. Total activity refers to activity measured in the presence of 2 r g of CaM/mL under standard assay conditions.
160
120
-
It \
1
-
0,
L
C
0
0 80
80
-
s 40
60 A
0 K
+
0
z
2
4
6
8
1 0
1 2
mol A r g / m o l c a t a l y t i c s u b u n i t
0 40
s 20
0
FIGURE 5: Protective effect of CaM on the loss of CaM-stimulated activity by pHPG treatment. Enzyme (68.2 pg/mL) was incubated in the presence of 40 mM pHPG, 6% (v/v) glycerol, and 250 pM calcium with varying amounts of CaM (0-22.2 pg). Incubation mixtures were diluted 10-fold, and 50-pL aliquots were assayed for phosphodiesteraseactivity at 2 mM cyclic AMP. Activity is expressed as percent of CaM-stimulatedcontrol activity incubated in the absence of pHPG.
Inactivation of CaM stimulation could be prevented by the inclusion of calcium and CaM in modification mixtures containing pHPG (Figure 5). The degree of protection was dependent upon the amount of CaM present during pHPG treatment and was maximum at a concentration slightly above
FIGURE 6:
Inactivation of CaM-stimulated phosphodiesterase as a function of [7-14C]phenylglyoxalincorporation. Enzyme (91.87 pg/mL) was reacted with 5 mM radiolabeled phenylglyoxal ( 2 5 mCi/mmol) in the presence of 13.3%(v/v) glycerol for various time periods. Enzyme activity was measured after treatment with phenylglyoxal by dilution of the enzyme into assay mixtures. Duplicate samples were terminated by addition of arginine as described under Materials and Methods and dialyzed for 48 h, rinsed from dialysis sacks with 0.5 mL of 0.1% Triton X-100, and counted to determine dpm. Control mixtures without enzyme were used to measure the completeness of dialysis.
a 1:l molar ratio of CaM to the phosphodiesterase catalytic subunit. Maximum protection was about 85% of the control CaM-stimulated activity under these conditions. The possibility that chemical modification of the enzyme by pHPG caused conversion to a CaM-independent, 4.1s monomeric enzyme form (Ahluwalia et al., 1984) was investigated. However, after treatment of the enzyme with 40 m M pHPG for 35 min, the Stokes radius as determined by gel filtration (data not shown) was similar to the untreated enzyme. The sedimentation coefficient estimated by density gradient centrifugation was also unchanged after pHPG treatment. The relationship between arginyl modification as measured by ['4C]phenylglyoxal incorporation and the inactivation phase of CaM-stimulated activity is presented in Figure 6. Similar experiments with different enzyme preparations indicated that
2216
BI OCH E M ISTR Y
approximately five to seven residues were modified per subunit on the basis of a 2:l reaction stoichiometry of PG to arginyl residue (Takahashi, 1977). Amino acid analysis of untreated and phenylglyoxal-modified phosphodiesterase also demonstrated that approximately 7 of the 27 arginyl residues estimated to be present per catalytic subunit of average M , 61.5 K were modified after 20 min of reaction with 5 mM PG. Lysyl residues were not found to be modified by these treatment conditions. Other arginine-modifying reagents, 2,3-butanedione and 1,2-~yclohexanedione,were not effective in promoting inactivation of CaM-stimulated activity. Butanedione inhibited CaM stimulation by only 25% after 60 min of treatment. The possible reaction of PG and pHPG with lysine residues was considered unlikely due to the inability of 2 mM pyridoxal phosphate or 20 mM ethyl acetimidate to alter CaM-stimulated activity (data not shown) and the direct results of amino acid analysis. DISCUSSION Treatment of CaM-dependent cyclic nucleotide phosphodiesterase under mild conditions with chemical reagents possessing specificity for arginine resulted in a complex activation-inhibition pattern suggesting two reactive classes of guanidyl groups. Enhancement of enzyme activity by chemical modification has been observed previously (Plapp, 1970; Shimizu, 1979). Further, arginine modification has been shown to selectively interfere with allosteric activation of an enzyme without affecting its catalytic activity (Carlson & Preiss, 1982). The transient activation phase observed during modification of phosphodiesterase appears related to the enhancement of CaM-stimulated activity by NH4Cl and imidazole (Klee et al., 1979) since this activation phase was not seen in later supplemental experiments where assays of control and treated enzyme were performed in the presence of 200 mM NH4C1. After this transient activation phase, the rate of loss of CaM-stimulated activity followed pseudo-first-order inactivation kinetics. Both the affinity for CaM and the extent of CaM activation appear affected by modification based on CaM activation curves. A thorough kinetic study of the modified enzyme will better define these alterations in enzyme activity. The selective effect of arginine modification on the inactivation of CaM-stimulated activity was dependent upon the conditions of modification since high concentrations of PG (>20 mM) led to a loss in basal activity when treatment was extended beyond 20 min. PG was less selective than pHPG in its action on lysophosphatidylcholine-stimulated activity as well. The efficacy of the glyoxal derivatives in promoting selective inactivation may be related to their polarity if the critical arginine residues are associated with hydrophobic regions of the enzyme. Steric factors associated with modification do not appear to be involved since pretreatment of the enzyme with butanedione in bicarbonate buffer had no substantial effect on inactivation by PG. That changes in CaMstimulated activity are due to chemical modification of the enzyme is supported by incorporation of radiolabeled phenylglyoxal and amino acid analysis of the untreated and modified enzyme. Both procedures indicated that seven arginines per catalytic subunit with an average of 61 500 kDa are selectively modified over the course of the observed changes in CaM-stimulated activity. These values can only be regarded as approximate since the affinity-purified enzyme was still heterogeneous and contained significant contaminating proteins as judged by specific activity and gel electrophoresis. Further, the results of this study must be interpreted with added caution since the enzyme preparation is composed of a mixture of
N I B H A N U P U D Y ET A L .
CaM-dependent isozymes of 63 and 60 kDa as indicated above. The susceptibility and sensitivity of each isozyme may not be the same since the subunits, although sharing certain immunological determinants, appear structurally distinct by peptide mapping (Sharma et al., 1984). Chemical modification of individual isozymes will be required to assess this possibility. Phospholipids activate CaM-deficient phosphodiesterases to relatively the same extent as CaM (Wolff & Brostrom, 1976; Pichard & Cheung, 1977); however, the simultaneous effect of both agents is neither additive nor synergistic. The persistence of lysophosphatidylcholine stimulation during pHPG treatment suggests that the catalytic domain of the modified enzyme remains functional and that phospholipid and CaM act by distinctly different mechanisms in promoting activation of the enzyme. Limited proteolysis of CaM-deficient enzyme causes an irreversible activation with an associated loss of CaM dependence (Kincaid et al., 1985). Chemical modification by pHPG would be expected to directly alter susceptibility to trypsin action; however, since activation of the pHPG-modified enzyme by different protease treatments was nearly the same, the possibility that specific chemical modification of an “inhibitory domain” results in a decreased susceptibility to general proteolytic activation merits further study. The inability of arginine modification of CaM to alter its function in stimulating phosphodiesterase is supported by previous investigations with other reagents (Tanaka et al., 1983; Walsh & Stevens, 1977). By contrast, modification of the carboxyl groups of CaM causes a loss in activity (Walsh & Stevens, 1977). Thus, certain acidic residues on CaM may be necessary for electrostatic interaction with basic groups at the recognition site of target proteins, leading to stabilization of complementary hydrophobic contacts. Recent studies (King & Heiny, 1987) indicate that phenylglyoxal treatment of the CaM-stimulated phosphatase calcineurin resulted in modification of arginyl residues essential for catalytic activity. In a target enzyme such as phosphodiesterase where basal catalytic activity is not altered, correlation between selective [ ’‘C]phenylglyoxal labeling of peptide fragments and high-affinity binding to CaM may be a useful approach to the characterization of the CaM recognition sites displaying an interactive diversity. REFERENCES Ahluwalia, G., Rhoads, A. R., & Lulla, M. (1984) Znt. J . Biochem. 16, 483-488. Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Butcher, R. W., & Sutherland, E. W. (1962) J . Biol. Chem. 237, 1244-1 250. Cachia, P. J., Van Eyk, J., Ingraham, R. H., McCubbin, W. D., Kay, C. M., & Hodges, R. S. (1 986) Biochemistry 25, 3553-3562. Carlson, C . A., & Preiss, J. (1982) Biochemistry 21, 1929-1934. Cheung, S.-T., & Fonda, M. L. (1979) Biochem. Biophys. Res. Commun. 90, 940-947. Cox, J. A., Comte, M., Fitton, J. E., & DeGrado, W. I. (1985) J . Biol. Chem. 260, 2527-2534. Hanley, R. M., Means, A. R., Ono, T., Kemp, B. E., Burgin, K. E., Waxham, N., & Kelly, P. T. (1987) Science (Washington, D.C.)237, 293-297. Kakiuchi, S., Sobue, K., Yamazaki, R., Kambayashi, J., Sakon, M., & Kosaki, G. (1981) FEBS Lett. 126, 680-685.
Biochemistry 1988, 27, 221 7-2222 Kincaid, R. L., Stith-Coleman, I. E., & Vaughan, M. (1985) J . Biol. Chem. 260, 9009-9015. King, M. M., & Heiny, L. P. (1987) J . Biol. Chem. 262, 10658-1 0662. Klee, C. B., Crouch, T. H., & Krinks, M. H . (1979) Biochemistry 18, 722-729. LaPorte, D. C., Toscano, W. A., Jr., & Storm, D. R. (1979) Biochemistry 18, 2820-2825. LaPorte, D. C., Wierman, B. M., & Storm, D. R. (1980) Biochemistry 19, 3814-3819. Levy, H. M., Leber, P. D., & Ryan, E. M. (1963) J. Biol. Chem. 238, 3654-3659. Malencik, D. A., & Anderson, S. R. (1 983) Biochemistry 22, 1995-2001. Manalan, A. S., & Klee, C. B. (1984) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 18, 227-278. McDowell, L., Sangal, G., & Prendergast, F. G. (1985) Biochemistry 24, 2979-2984. Newton, D. L., Oldewurtel, M. D., Krinks, M. H., Shiloach, J., & Klee, C. B. (1984) J. Biol. Chem. 259, 4419-4426. O'Neil, K. T., & DeGrado, W. F. (1985) Proc. Natl. Acad. Sci. U.S.A.82, 4954-4958.
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Pichard, A.-L., & Cheung, W. Y . (1977) J. Biol. Chem. 252, 4872-4875. Plapp, B. V. (1970) J . Biol. Chem. 245, 1727-1735. Rhoads, A. R., Lulla, M., Moore, P. B., & Jackson, C. E. (1985) Biochem. J. 229, 587-593. Sharma, R. K., Wang, T. H., Wirch, E., & Wang, J. H. (1980) J . Biol. Chem. 255, 5916-5923. Sharma, R. K., Taylor, W. A., & Wang, J. H. (1983) Methods Enzymol. 102, 2 10-2 19. Sharma, R. K., Adachi, A.-M., Adachi, K., & Wang, J. H. (1 984) J . Biol. Chem. 259, 9248-9254. Shimizu, T. (1979) J . Biochem. (Tokyo) 85, 1421-1426. Takahashi, K. (1977) J . Biochem. (Tokyo) 81, 395-402. Tanaka, T., Ohmura, T., & Hidaka, H. (1983) Pharmacology 26, 249-257. Walsh, M., & Stevens, F. C. (1977) Biochemistry 16, 2742-2745. Weiss, B., Prozialeck, W. C., & Wallace, T. L. (1982) Biochem. Pharmacol. 31, 22 17-2226. Wolff, D. J., & Brostrom, C. 0. (1976) Arch. Biochem. Biophys. 173, 720-73 1.
Inactivation of Escherichia coli Pyruvate Formate-Lyase by Hypophosphite: Evidence for a Rate-Limiting Phosphorus-Hydrogen Bond Cleavage? Edward J. Brush, Koren A. Lipsett, and John W. Kozarich*J Department of Chemistry and Biochemistry and Agricultural Biotechnology Center of the Maryland Biotechnology Institute, University of Maryland, College Park, Maryland 20742 Received October 29, 1987; Revised Manuscript Received January 5, 1988
J., Neugebauer, F. A,, Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Nutl. Acad. Sci. U.S.A.81, 13321 have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vm,,/Km isotope effect of 3.6 f 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction. ABSTRACT: Recently, Knappe and co-workers [Knappe,
P y r u v a t e formate-lyase (EC 2.3.1 S 4 ; formate acetyltransferase; PFL)] catalyzes the key reaction in anaerobic glucose metabolism-the conversion of pyruvate and coenzyme A (CoA) to acetyl-coA and formate (Scheme I). The enzyme is a homodimeric protein of M , 170000 which occurs in both 'This research was supported by Research Grant GM 35066 from the National Institute of General Medical Sciences, US. Public Health Service. 'American Cancer Society Faculty Research Awardee ( 1 983-1 988).
0006-2960/88/0427-22 17$01.50/0
an inactive and active form (Knappe et al., 1969, 1974). The activation process is catalyzed under conditions of anaerobiosis by an Fe(I1)-dependent activating enzyme of M , 30000 (Knappe et al., 1969). In vitro activation also requires py-
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Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; EPR, electron paramagnetic resonance; PFL, pyruvate formate-lyase; SAM, S-adenosylmethionine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris, tris(hydroxymethy1)aminomethane.
0 1988 American Chemical Society
