Mechanism of Phosphatidylinositol-Specific Phospholipase C

Chapter 7

Mechanism of Phosphatidylinositol-Specific Phospholipase C Revealed by Protein Engineering and Phosphorothioate Analogs of Phosphatidylinositol

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 16, 2018 | https://pubs.acs.org Publication Date: December 15, 1998 | doi: 10.1021/bk-1999-0718.ch007

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Robert J. Hondal , Zhong Zhao , Alexander V. Kravchuk , Hua Liao , Suzette R. Riddle , Karol S. Bruzik , and Ming-Daw Tsai 2

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Department of Chemistry, Ohio State Biochemistry Program, and Department of Biochemistry, The Ohio State University, Columbus, OH 43210 The catalytic mechanism of phosphoinositide-specific phospholipase C was investigated by the combined use of protein engineering and application of phosphorothioate analogs of phosphatidylinositol as substrates. The results showed that three residues: His32, His82, and Arg69, each contribute to catalysis up to 10 -fold rate acceleration factor. In addition, the carboxylic acid residue of Asp274 also contribute to catalysis by interacting with His32 to form a general base, whereas Asp33 forms a triad with Arg69 and His82 which function as general acid both activating the phosphate group for nucleophilic attack and assisting the leaving group. The overall mechanism can be described as involving a complex general acid-general base catalysis. 5

From an enzymologists' point of view, the mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC, E C 3.14.10) is of great interest because of its close similarity to the mechanism ofribonucleaseA , an enzyme which has been studied for more than 35 years (1-3). A general base-general acid (GB-GA) mechanism for ribonuclease A involving His 12 and His 119, respectively, has been widely accepted by most enzymologists. However, the "classical" G B - G A mechanism of ribonuclease A has been the subject of controversy in recent years because of the proposal by Breslow (4) in which he suggested that the role of His 119 is to first protonate the nonbridging oxygen of the phosphate group to yield a "triester-like" species. The picture of catalysis was further complicated by Raines and coworkers (5) who have proposed a catalytic role for Lys41 via a hydrogen bonding to a nonbridging oxygen atom of the phosphate group. Gerlt and Gassman have termed this type of hydrogen bonding interaction a low barrier hydrogen bond (6). In summary, a complete catalytic site of RNase A is comprised of three elements, G B , G A , and the phosphate stabilizing residue. PI-PLC catalyzes the conversion of phosphatidylinositol (PI) to 1-inositol phosphate (IP) in two distinct steps, via the formation of 1,2-cyclic phosphate (IcP) and its hydrolysis to inositol 1-phosphate as shown in Figure 1. The first transesterification step is ca. 10 -fold faster than the second hydrolysis step (7). The 4 3

4 C u r r e n t address: Department of Medicinal Chemistry and Pharmacognosy, College of Phar macy, University of Illinois at Chicago, Chicago, IL 60612

5Corresponding author. ©1999 American Chemical Society

Bruzik; Phosphoinositides ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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110 results of our earlier stereochemical studies using phosphorothioate and oxygenisotope labeled analogs (8,9) demonstrated that bacterial PI-PLC catalyzes two S 2type reactions resulting in the overall retention of configuration at the phosphorus atom, which immediately suggested a mechanism similar to that of ribonuclease A (70). Consistently, the recent x-ray structure of PI-PLC complexed with myoinositol revealed the presence of His32 and His82 at the active site (77). These two histidine residues are almost superposable on the two histidines of the ribonuclease A active site (77), and are likely to perform the analogous functions. Both enzymes, PI-PLC and ribonuclease A, catalyze the conversion of a phosphodiester to a cyclic intermediate via intramolecular attack of a β-hydroxyl group, followed by slow hydrolysis of the fivemembered cyclic product to a linear phosphomonoester. In the case ofribonucleaseA , the first transphosphorylation step produces the 2',3'-cyclic nucleotide, which is released (12,13). Analogously to the hydrolysis of IcP, the cyclic phosphodiester is then only slowly hydrolyzed to form a 3'-phosphomonoester. The initial use of site-directed mutagenesis to study the mechanism of PI-PLC (14,15) has largely confirmed the catalytic mechanism proposed based on the x-ray structure (77) and stereochemical results (8,9), and its similarity to that of ribonuclease A . The pair of active-site histidines was found absolutely essential for catalysis, and Arg69 was proposed to play the role of the third element analogous to that of Lys41 in ribonuclease A (5,11,14,15). Since both enzymes catalyze an intramolecular phosphate-transfer reaction, it is perhaps not surprising that they operate by using analogous chemical mechanisms. It is remarkable however, that both enzymes have evolved to utilize homologous catalytic machineries, when one is a phospholipase that works at a water-lipid interface, and the other is a hydrolytic nuclease. The results of S D M obtained for PI-PLC allowed definition of the catalytically important residues, however by themselves they did not allow drawing detailed mechanistic inferences. A more precise picture of the mechanism was obtained by application of the combined approach using SDM, and kinetic and stereochemical analysis of phosphorothioate substrate analogs (14,16-18). The results obtained in this way for PI-PLC could also be significant to the mechanism ofribonucleaseA , due to similar topology of the two active sites. This review summarizes our work on PIP L C to date (14,16-18), and compares and contrasts the mechanisms of these two enzymes.


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Site-Directed Mutagenesis In addition to the presence of His32 and His82, the x-ray structure of PI-PLCinositol complex revealed that each histidine is in close relationship with the carboxyl group of Asp274 and Asp33, respectively. It has been previously shown that His-Leu mutations completely abolished activities in mutants, indicating that these two histidines are essential for catalysis (77). It has been further demonstrated that the mammalian PI-PLC also uses homologous histidines for catalysis (19,20). Using site-directed mutagenesis, we have mutated the two active site histidines to alanine residues (18). The results of specific activity assay using H-PI showed that His32 and His82 each contribute a factor of ca. 10 toward catalysis (Table 1). Likewise, the carboxylic residues of Asp274 and Asp33 also contribute sigmiicantly toward catalysis. We have found that the D274A mutant is 10 -fold less active than WT PIPLC, whereas the D274N mutant retains significant activity. The latter finding can be explained by the fact that asparagine is isosteric with aspartate and can maintain a similar hydrogen bonding pattern. Both the structure (77) and SDM data (14,15,18) indicate mat Asp274 and His32 function together with the 2-OH group of inositol as a "catalytic triad" similar to those of serine proteases (27). These results are consistent with the proton-relay function of His32«-Asp274. In contrast to Asp274, the mutation of Asp33 to alanine decreased the catalytic rate only by a factor of 10 , and unlike the D274N mutant, the D33N mutant had only slightly higher activity than D33A (14,18). 3

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Finally, mutation of Arg69 to alanine caused ca. 10 -fold decrease in activity with respect to WT, while mutation to lysine lowered the rate 10 -fold (14). Thus, the results of SDM described so far indicated that His32, His82, and Arg69 each contribute a factor of 10 toward catalysis, however their specific function in the catalytic machinery remained somewhat unclear. 3

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Use of Phosphorothioates to Study Active Site Interactions Substitution of a nonbridging oxygen of a phosphate group with sulfur has been a widely used approach to study enzyme mechanisms, in view of the fact that phosphorothioate diesters bear a stereogenic phosphorus atom, and can be used to investigate steric course of the displacement reactions at phosphorus. These phosphorothionates are chemically less reactive than phosphates, and display also slower rates of enzymatic cleavage reaction (23). The rate decrease upon O/S modification is commonly referred to as the "trûo-efïect" (classified as the Type I thioeffect). In contrast to phosphorothionates, substitution of a bridging oxygen by a sulfur atom produces a phosphorothiolate which is chemically more reactive than the corresponding phosphate. The difference in reactivity obtained upon such modification is referred to as the Type Π thio-effect. It is worth mentioning that despite rather large rate effects in enzymic reactions, the O/S modification can be regarded as a minor one from the structural point of view, allowing exacdy the same binding modes of phosphorothioates as compared to phosphates. Thus, given the differential ability of oxygen and sulfur to act as hydrogen bond acceptors (24), the combination of SDM and phosphorothioate analogs offers a unique possibility of examining interactions of specific enzymic residues (such as e.g. Arg69) with specific sites of the substrate (such as the nonbridging/bridging oxygen/sulfur atom). In order to get further insights into the catalytic mechanism of PI-PLC we have employed both types phosphorothioate analogs of phosphatidylinositol to study reactions catalyzed by mutants altered at Arg69, Asp33 and His82 positions. The results obtained from this approach provide far more detailed insights into the enzyme mechanism than application of S D M or substrate alteration alone. Role of Arg69 in Catalysis: Nonbridging Thio-Effects. Substitution of the nonbridging oxygen atom with sulfur results in formation of a pair of diastereomers of the phosphorothioate analog of PI, designated (/? )-DPPsI and (5)-DPPsI (Figure 2). The /? -isomer has been previously used in our investigation of the steric course of PIP L C catalyzed formation of IcP (8). This isomer is turned over by the enzvme only slightly more slowly than the natural substrate (k /k = 3) (Table 2). The Ρ N M R time course of the reaction of the 1:1 mixture of both diastereomers with the WT PIPLC revealed that this enzyme prefers the /? -diastereomer over the S -DPPsI isomer by a factor of 1.6 χ 10 . To the best of our knowledge, this is the largest enzymic stereoselectivity observed to date between phosphorothioate diastereoisomers. Another important finding was that the R69K mutant showed 10 -times lower stereoselectivity, k / k = 16 (14). This great relaxation of stereoselectivity is achieved by the mutant enzyme by maintaining the same rate of conversion of the 5 -isomer as the WT enzyme, while reducing activity toward the /? -isomer by a factor of 10 -fold. This result is consistent with the PI-PLC crystal structure showing that Arg69 is in the correct position to stabilize the negative charge of the phosphate group in the transition state (77). The extremely high stereoselectivity observed with the WT enzyme, and the great relaxation of stereoselectivity observed with the R69K mutant, is an unambiguous evidence for a strong direct interaction between the guanidinium side chain of Arg-69 and the pro-S oxygen of the phosphate group. Assuming tentatively that there is no hydrogen bonding stabilization between Arg69 and the sulfur atom in p

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RCOO RCOOPI-PLC fast Λ > DAG κ


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.P»0 O^ L^-r-OH HOA^r^OH OH IcP

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PI-PLC slow H0 '

l / H0 x *»o U^-T-OH ΗΟΛ^-τΛ^ΟΗ OH p

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Figure 1. Reactions catalyzed by bacterial phospholipase C.

phosphatidylinositol-specific

Table 1. Summary of Kinetic Data for WT and Mutant PI-PLC. 3

DPsPI

H-PI

Specific Activity (μπιοί min" mg ) 1300

V i 1 (μπιοί min mg ) 53.5

H32A

0.0301

Mechanism of Phosphatidylinositol-Specific Phospholipase C

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