Kinetics and mechanism of angiotensin phosphorylation by the transforming gene product of Rous sarcoma virus.

We have studied steady state kinetics of phosphorylation of [Val5]angiotensin II by pp60src, the transforming gene product of Rous sarcoma virus. Results of initial rate studies at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 mM, respectively, and Vmax was 1.0 nmol/min/mg. The end product ADP and the ATP analog AMP-PNP were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations, but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. We also presented evidence that, while pp60src contained essential histidine and/or lysine residues in its active site, the mechanism does not involve a phosphoryl enzyme intermediate.


Kinetics and Mechanism of Angiotensin Phosphorylation by the Transforming Gene Product of Rous Sarcoma Virus*
(Received for publication, August 19,1983) Tai Wai Wongz and Allan R. Goldberg From The Rockefeller University, New York, New York 10021 We have studied steady state kinetics of phosphorylation of [Vals]angiotensin I1 by pp60"'", the transforming gene product of Rous sarcoma virus. Results of initial rate studies at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 W M and 0.24 mM, respectively, and V,,, was 1.0 nmol/min/mg. The end product ADP and the ATP analog AMP-PNP were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations, A dead-end analog of angiotensin 11, [APhe4]angiotensin 11, was a noncompetitive inhibitor at varying ATP concentrations, but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. We also presented evidence that, while pp60"'" contained essential histidine and/or lysine residues in its active site, the mechanism does not involve a phosphoryl enzyme intermediate. Tyrosyl protein kinase activity has been demonstrated to be integral to the transforming gene products of several different retroviruses, to be associated with cellular receptors for growth factors, and to be present as cytoplasmic components in normal cells (1)(2)(3)(4)(5)(6). The introduction of small peptide substrates for tyrosyl protein kinases has facilitated progress in understanding the functions and properties of these enzymes (7)(8)(9). These peptides are either fragments of a viral transforming protein or angiotensin analogs. The former group of peptides has amino acid sequences that include the in uivo tyrosine phosphorylation site. The latter group of peptides, however, has as yet no obvious relation to t h e enzymes in uiuo. Nonetheless, their usefulness has been demonstrated in studies of viral protein kinases as well as in identification of novel enzymes in normal rat liver (6,9).
A great number of reports have dealt with characterization of the tyrosyl protein kinase activity associated with the transforming protein, pp60"'", of Rous sarcoma virus (10).
However, none of these reports have addressed the question of mechanism whereby in uitro phosphorylation proceeds.
Here we describe results of kinetic experiments in which we * This work was supported in part by United States Public Health Service Grants CA13362 and CAB213 and by Biomedical Research Support Grant SO7 RR07065. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ angiotensin I1 at various concentrations. Incubation was at 30 "C for 25 min, followed by 3 min at 90 "C. Peptide phosphorylation was assayed by paper electrophoresis as described previously (9) except that electrophoresis buffer also contained 5 mM EDTA.
Data Processing-The nomenclature of Cleland was used in designation of reactants and kinetic constants (12). All experiments were performed at least three times, and the results presented here are representative of each experiment. Initial rate data were fitted by linear regression analyses to the rate equation described in Scheme l a for a sequential mechanism. Intercepts and slopes of double reciprocal plots were replotted against l/[substrate] or [inhibitor] to obtain V,,,,,, Michaelis constants, dissociation constants, and inhibition constants according to relationships described by Cleland (12,13).

RESULTS
We previously reported the use of angiotensin analogs as in uitro substrates for tyrosyl protein kinases (9). We have now used [Val'langiotensin I1 in studying the kinetics and mechanism of phosphorylation catalyzed by pp60"", the transforming gene product of Rous sarcoma virus. The enzyme was partially purified from Rous sarcoma virus-transformed rat cells; it was free of serine and threonine kinase activity and showed a linear time course of phosphorylation over 30 min (Ref. 9 and data not shown). Rate of phosphorylation of [Val5] angiotensin I1 increased with increasing concentration of peptide up to about 1.5 mM (Fig. la, open circles). At concen-  Reaction rates were also determined at 1 pM ATP (0) and were indicated by the right-hand ordinate. b, reaction rates were determined at 2 m M peptide. Peptide phosphorylation was assayed as described under "Experimental Procedures." (above 4 mM) seemed to be inhibitory ( Fig. l a , closed circles). The rate of reaction also was linear with respect to ATP concentration up to at least 180 pM (Fig. lb). Initial rates were determined by varying concentration of one substrate while holding concentration of the other substrate fixed, and results are expressed in double reciprocal plots. When the concentration of ATP was varied, increasing concentration of AT I1 resulted in a decrease in both the slope and the intercept of the plot (Fig. 2u). The same effect was evident with fixed concentrations of ATP and varying concentrations of peptide (Fig, 2%). In both cases, the lines converge to the left of the ordinate. These patterns are indicative of a sequential mechanism and rule out a Ping Pong Bi Bi pathway, in which case one would have observed parallel plots.
We examined the effects of one of the end products, ADP, on the reaction rates. At fixed concentration of peptide and varying concentrations of ATP, increasing concentrations of ADP caused a change in the slope, but not the intercept, of the double reciprocal plots (Fig. 3u). At fixed concentrations of peptide, both slope and intercept increased with increasing concentrations of ADP (Fig. 3b). ADP is therefore a linear competitive inhibitor of ATP and a linear noncompetitive  inhibitor of peptide. These results are consistent with a sequential mechanism in which ADP and ATP bind the same enzyme form. We have not succeeded in obtaining the other end product, phosphopeptide, in sufficient quantity to perform the same kind of analyses as were done with ADP. In order to differentiate between a random and ordered pathway, we sought to study the effects of dead-end inhibitors of either substrate. The analog AMP-PNP is a well characterized inhibitor of ATP (14) and was used for this purpose. As shown in Fig. 4, this analog was a competitive inhibitor of ATP and showed noncompetitive inhibition with peptide. These observations allowed us to rule out an Ordered Bi Bi or Theorell-Chance mechanism in which peptide binds first and ATP binds second. In those cases, uncompetitive inhibition would have been observed with varying peptide concentration. Data obtained from initial rate studies were replotted to yield Michaelis and dissociation constants for substrates and inhibition constants for product and analog inhibitors ( Table I). KnZ for ATP, as determined from replot of data in Fig. 2b, was 7.0 p~. However, data in Fig. I b suggest that the K , should be considerably higher than 7 p~. The discrepancy may be a result of inhibition by peptide substrate (Fig. la and  see below). Data in Fig. 2b and Table I were determined at  TABLE I relatively low concentrations of peptide substrate whereas t.hose in Fig. 16 were obtained with 2 mM peptide. Under the latter conditions, some degree of inhibition by peptide may have been taking place, and a higher concentration of ATP was therefore required to reach the half-maximal rate. Also, with peptide as varying substrate, Kii for ADP is severalfold higher than Kk. The latter constant approximates the dissociation constant for enzyme-ADP complex, K,. The difference between Kii and K, may be due to the fact that concentration of ATP used in the experiment was close to K , of ATP. Hence, the apparent K;; is in effect close to being twice the value of K , (Kt, being equal to K, multiplied by (1 + A/K,,)).
If this factor is taken into account, Kii would then be comparable to K,.
We next examined a number of angiotensin analogs for their effects on phosphorylation of AT 11. Three of the peptides examined had the tyrosine of AT I1 replaced with a different residue. None of the peptide analogs was phosphorylated by pp60"".* Surprisingly, only one of the five peptides, the dehydrophenylalanyl analog of AT 11, acted as an inhibitor ( Table 11). The other peptides had no observable effect on the reaction rate at concentrations up to 5 mM (not shown). When [3Phe4]AT I1 was used in initial rate studies, it was found to result in parabolic noncompetitive inhibition with ATP (Fig. 5a). However, a rather complex pattern of double reciprocal plots was obtained when ATP concentration was held constant and peptide concentration was varied (Fig. 56).

T. W. Wong and A. R. Goldberg, unpublished observations.
In the presence of inhibitor, the reaction rate increased with increasing concentration of peptide up to about 0.16 mM. Beyond that concentration, the reaction rate decreased asymptotically. The noncompetitive inhibition by [APhe4JAT I1 at varying ATP concentrations ruled out a simle Ordered Bi Bi or Theorell-Chance mechanism with ATP as the first binding substrate. Thus, we have to differentiate between a rapid equilibrium Random Bi Bi mechanism or a modified Ordered Bi Bi mechanism in which the free enzyme binds peptide, but the binary complex does not allow subsequent binding by ATP. A schematic representation of the latter kinetic mechanism is shown in Fig. 6a. Such a pathway resembles a rapid equilibrium Random Bi Bi reaction in that the free enzyme binds both substrates. However, a productive sequence would ensue only if ATP binds enzyme first, followed by binding of peptide to the binary complex. Binding of peptide to the free enzyme may result in steric hindrance that precludes ATP from access to the active site. In the model depicted in Fig. 6a, substrate inhibition at high peptide concentration is most likely due to formation of dead-end complex EB, and perhaps also of EB2. At high ATP concentration (40 p M or greater), the enzyme preferentially cycles between E and EA by virtue of mass action, and inhibition by peptide is alleviated. Results in Fig. 56 suggest that the presence of [APhe4]AT I1 induced substrate inhibition at concentrations of AT I1 that by itself would not have been sufficient to cause inhibition. A mechanism that would account for the induced substrate inhibition is depicted in Fig.  6b. In such a pathway, the binding of inhibitor results in a conformational change in the enzyme such that a second molecule of peptide substrate or inhibitor will be accommodated. The additional equilibria that have to be considered are  61 + d 2 ) . At varying concentrations of AT 11, the equation is of the form y = a/x + x / b and predicts a hyperbolic concave-up pattern for the double reciprocal plot. Furthermore, the equation in Scheme IC predicts that substrate inhibition requires the presence of I. Thus, it appears that the data obtained could be adequately interpreted by the Ordered Bi Bi mechanism shown in Fig. 6. On the other band, a Random Bi Bi mechanism would not be compatible with the kinetic data, especially with the inhibition pattern obtained with [APhe4]AT 11. We replotted the slopes and intercepts of Fig, 5a uersus concentrations of peptide inhibitor. By doing that, we obtained parabolic curves for both intercept and slope replots (not shown), which were extrapolated to yield apparent Ki2 and K , of 0.8 and 0.6 mM, respectively. We did not attempt to obtain sufficient data to calculate the various equilibrium constants for the enzyme-inhibitor complexes. SCHEME 1. Proposed rate equations for sequential bireactant reaction and for phosphorylation of AT 11. a, rate equation for a sequential bireactant mechanism. A and B are concentrations of substrates; K, and Kb are Michaelis constants for substrates A and B; and K, is dissociation constant for the complex EA. b, rate equation for mechanism depicted in Fig. 66. Z stands for concentration of [APhe'IAT I1 and K values are equilibrium constants for enzymeinhibitor complexes as described in the text. c, double reciprocal form Results of the kinetic experiments suggest that the in vitro phosphorylation of angiotensin I1 by pp60"" proceeds via a ternary complex intermediate. However, there is as yet no evidence to exclude the possibility that a phosphorylated enzyme form mediates the phosphoryl transfer from ATP to the phenolic moiety of peptide. It is possible that phosphoryl transfer occurs between ATP and enzyme, but ADP does not depart from the active site until after the transfer between phosphoryl enzyme and AT I1 has taken place. We examined this possibility by studying the effects of chemical modification on the enzymatic activity. Diethyl pyrocarbonate reacts with imidazole and amino groups and has been used to selectively modify histidine residues of enzyme active sites (15). Before initiating kinase reaction, we preincubated reaction mixtures with various concentrations of diethyl pyrocarbonate. As shown in Fig. 7, the enzymatic activity was inactivated by 50% by 0.08% diethyl pyrocarbonate and was almost abolished at concentration above 0.15%. Preincubation of the reaction mixture in the presence of ATP did not provide any significant protection of the enzyme. These observations suggest that if a phosphoryl enzyme intermediate existed, the modified residues would not likely have been imidazole or amino moieties. This is because in those cases phosphorylation of enzyme should have provided some protection from inactivation by diethyl pyrocarbonate. We cannot rule out the possibility that formation of phosphoryl enzyme intermediate requires the presence of peptide substrate. However, experimental verification of that possibility is not presently feasible. Furthermore, we re-examined the phosphoamino acid content of pp60"" labeled in uitro with [T-~'P]ATP. In previous studies, analyses were performed with acid hydrolysates. Under those conditions, phosphoamino acids other than phosphoserine, phosphothreonine, and phosphotyrosine would have been completely destroyed. We therefore performed base hydrolyses on samples of 32P-labeled pp60"" and separated the hydrolysates by two-dimensional thin layer electrophoresis under conditions that allowed separation of phosphoserine, phosphothreonine, phosphotyrosine, 3-phosphohistidine, and phosphoarginine. Again, phosphotyrosine was the only radiolabeled phosphoamino acid observed.' Since Snyder et al. (16) observed previously that Rous sarcoma virus mutants lacking the tyrosine phosphorylation site exhibited unabated protein kinase activity, our data prompted us to conclude that I I % DEP FIG. 7. Inactivation of pp60"" kinase activity by diethyl pyrocarbonate. Enzyme (400 ng) was preincubated at pH 6.5 at 30 "C for 5 min in the absence (0) or presence (A) of 40 P M ATP. Diethyl pyrocarbonate (DEP) at various concentrations was then added to the reaction mixtures, and incubation was continued for 10 min. Bovine serum albumin was then added to 0.1 mg/ml, and aliquots were removed for assaying kinase activity. the reaction mechanism probably does not involve a phosphoryl enzyme intermediate.

DISCUSSION
By studying steady state kinetics of the phosphorylation of angiotensin 11, we have determined the effects of co-substrates, product, and substrate analogs on the initial rate of reaction. The kinetic data allow us to conclude that the kinase reaction follows a sequential pathway. The order of substrate addition was determined by studying effects of AMP-PNP and [APhe4]angiotensin 11. The substrate inhibition induced by the presence of [ APhe4]angiotensin I1 was most helpful in the diagnosis of an Ordered Bi Bi mechanism. In the mechanism proposed, the enzyme binds either ATP or peptide. However, occupation of the active site by AT I1 precludes subsequent binding by ATP and therefore does not allow for a productive reaction. Thus, the mechanism can be viewed as intermediate between a rapid equilibrium Random Bi Bi and an Ordered Bi Bi mechanism.
The observation that most analogs of AT I1 did not inhibit peptide phosphorylation suggests that the enzyme exhibits rather stringent selectivity in binding substrate. [N02Tyr4] AT I1 was not phosphorylated by the enzyme presumably because the nitro group rendered the molecule too bulky for interaction with the active site. Similarly, other analogs were either too large (as in [Ain4]AT TI) or too small (as in AT I1 pentapeptide) to compete with AT I1 for binding sites. [APhe4] AT I1 differs from the other analogs in that the residue a t position 4 has a rigid planar geometry. This unusual feature of the peptide may distort the topology of the enzyme active site such that there will be sufficient room for a second molecule of peptide to bind. Occupation of the enzyme active site by two peptide molecules may account for inhibition at high concentration of peptide substrate. In the presence of [APhe4]AT 11, the process is facilitated, and inhibition is observed at much lower concentrations of peptide substrate. Substrate inhibition induced by a dead-end analog also was observed previously with yeast hexokinase and thymidylate synthetase (17, 18).
Results of our experiments also suggest that pp60"" contains essential histidine and/or lysine residues and that the reaction mechanism does not involve a phosphoryl enzyme.

21.
It remains to be determined if phosphorylation of that tyrosine residue of pp6OaW has any functional significance on the enzymatic activity. We have not been able to obtain sufficient quantity of phosphorylated AT I1 to study the reverse reaction: ADP + phosphopeptide = ATP + peptide. Fukami and Lipmann (19) previously demonstrated a similar reaction involving pp60"' " using anti-pp60"" IgG as substrate. Our results are almost identical with those obtained by Whitehouse et al. (20) for bovine heart and skeletal muscle CAMPdependent kinase using kemptide substrate. However, they differ in some aspects from those of Erneux et al. (21), who studied the steady state kinetics of phosphorylation of peptide substrate by epidermal growth factor-receptor kinase. The latter authors concluded that while the mechanism was also Ordered Bi Bi, the order of substrate binding was peptide first, and ATP second. At the moment, it is not clear if the apparent difference in our interpretations reflects any difference in enzymatic specificity.