P–O Bond Destabilization Accelerates Phosphoenzyme Hydrolysis of Sarcoplasmic Reticulum Ca2+-ATPase

The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm–1. This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 Å shorter terminal P–O bonds and a 0.09 Å longer bridging P–O bond that is ∼20% weaker, the angle between the terminal P–O bonds is wider, and –0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P–O bond of E2-P accounts for a 1011–1015-fold hydrolysis rate enhancement, implying that P–O bond destabilization facilitates phosphoenzyme hydrolysis. P–O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins. The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm–1. This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 Å shorter terminal P–O bonds and a 0.09 Å longer bridging P–O bond that is ∼20% weaker, the angle between the terminal P–O bonds is wider, and –0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P–O bond of E2-P accounts for a 1011–1015-fold hydrolysis rate enhancement, implying that P–O bond destabilization facilitates phosphoenzyme hydrolysis. P–O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins. Phosphorylation is one of the fundamental regulatory mechanisms in biology (1Mildvan A.S. Proteins. 1997; 29: 401-416Crossref PubMed Scopus (255) Google Scholar). It also governs the catalytic mechanism of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1) (2Hasselbach W. Makinose M. Biochem. Z. 1961; 333: 518-528PubMed Google Scholar), where it controls the ordered sequence of catalytic steps to ensure the efficiency of the overall catalytic reaction. Here we study one of the ATPase phosphoenzyme intermediates by monitoring an enzyme-catalyzed isotopic exchange reaction at the phosphate group with infrared spectroscopy. The result is an infrared spectrum at “atomic resolution” in a crowded spectral region. It observes selectively the transiently bound phosphate group, a group that cannot be studied by site-directed mutagenesis. Our approach can be extended to other phosphoproteins and reveals here the molecular cause of an important property of the phosphoenzyme studied. The Ca2+-ATPase (2Hasselbach W. Makinose M. Biochem. Z. 1961; 333: 518-528PubMed Google Scholar) pumps two Ca2+ ions against a concentration gradient across the sarcoplasmic reticulum membrane, which relaxes a flexed muscle (for reviews see Refs. 3Andersen J.P. Biochim. Biophys. Acta. 1989; 988: 47-72Crossref PubMed Scopus (99) Google Scholar, 4Hasselbach W. Bonting S.L. De Pont J.J.H.H.M. Membrane Transport. Elsevier, Amsterdam1981: 183-208Google Scholar, 5Inesi G. de Meis L. Martonosi A. 2nd Ed. The Enzymes of Biological Membranes. 3. Plenum Press, New York1985: 157-191Google Scholar, 6Martonosi A. Kracke G. Taylor K.A. Dux L. Peracchia C. Soc. Gen. Phys. Ser. Google Scholar, Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar, A. Biochem. PubMed Scopus Google Scholar). The for is by the which the ATPase at to at two and E2-P. The Ca2+ is with the from to E2-P. phosphoenzymes catalytic the phosphoenzyme with to the phosphoenzyme E2-P with This of catalytic the efficiency of the the of is that the by is Ca2+ is Ca2+ E2-P the model compound acetyl phosphate in The for E2-P hydrolysis is between and the PubMed Scopus Google Scholar, A. de Meis L. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, de Meis L. G. PubMed Google Scholar, W. Z. PubMed Scopus Google Scholar), that of acetyl phosphate is G. Soc. 1961; Scopus Google Scholar). This of the reaction by the is for the of the and for the of Ca2+ from the the of the phosphate group is important in the It has found to in the transition from to E2-P Meis L. Scopus Google Scholar, PubMed Scopus Google Scholar), and to for an hydrolysis rate of E2-P with Meis L. PubMed Scopus Google in Meis L. Biochim. Biophys. Acta. 1989; PubMed Scopus Google Scholar). The molecular mechanism of We were in the of the phosphate group controls catalytic and infrared spectroscopy to study the phosphate group of E2-P in spectroscopy has for the of protein C. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, W. Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. C. Biophys. PubMed Scopus Google Scholar) it of and bond at a of that that of and and of Biological New Scholar). the of infrared spectroscopy the of isotopic has to a group in a protein PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of the infrared of a group are shifted with to of the group and can be in the is to a group in the the of group be the of other the of protein in is the of infrared where the reaction of is in the infrared We a compound of from a for from valence A. W. W. PubMed Scopus Google in A. C. PubMed Scopus Google Scholar). 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Mg2+ bonds of and a bond of Å with in bond to Å is to the bond valence to and to for the in for E2-P. in bond are to the bridging P–O bond with a the catalytic of the of the we a shift of –0.2 formal charges from the group to the aspartyl moiety. formal charge is to bond the aspartyl of E2-P interactions with the that are from of the acetyl of acetyl The for the in interactions to be the two of the aspartyl which are to bonds in E2-P. The bonds to the of E2-P be somewhat of acetyl phosphate, which for valence of bond valence of see bond to the bridging is by the that the bond of the bridging to the has a bond valence of the bond is to a bond valence of in the of This is at with the of which that bonds to be the The in The Press, Scholar). the between the two the bridging a to which the bond valence of the with protein interactions with the bridging found for phosphate Biochem. 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, W. A. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar, C. L. PubMed Scopus Google Scholar). The to the aspartyl accounts for valence to one It to that the aspartyl phosphate of E2-P one bond acetyl phosphate in it is that the bonds to the aspartyl are bonds are shorter of interactions with of the aspartyl with ions is in E2-P –0.2 formal charges are shifted from the to the aspartyl moiety. The charge the oxygens can be by Mg2+ by which is to in the of the and the of the ATPase C. M. PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar). Mg2+ and a three of charge by of can bonds bond We suggest that Mg2+ and Lys684 with the aspartyl oxygens of E2-P. The of of Mg2+ to a in the distribution the phosphate The of the terminal phosphate oxygens of E2-P with of acetyl phosphate with Mg2+ to the valence The in The Press, Scholar) and a for an with bond the in formal charge the aspartyl oxygens in E2-P and a for it is that Mg2+ weaker bonds with phosphate oxygens and a bond with an aspartyl Mg2+ bridging a phosphate and an aspartyl of aspartyl phosphate can be in a protein is by the model compound acetyl phosphate the is Soc. Scopus Google Scholar). the reaction, of the ATPase by phosphate, an a for the and the reaction by 29: PubMed Scopus Google Scholar). for the of the E2-P the we a model for the interactions of the of which is in to model Mg2+ the and a terminal phosphate the of Lys684 with the bridging with the important of Lys684 in it was found that is for and of the phosphoenzyme to E2-P J.P. PubMed Google Scholar). was a structure of the ATPase was PubMed Scopus Google Scholar) that for a the here for bridging and a to the bridging Å are also found for other that an aspartyl phosphate the of PubMed Scopus Google Scholar), W. A. PubMed Scopus Google Scholar), and G. PubMed Scopus Google Scholar), with the two to the the of the Ca2+-ATPase L. Biochem. Scopus Google Scholar). bridging a Å from the bridging is observed for the C. L. PubMed Scopus Google Scholar). bridging a phosphate and the cannot the high hydrolysis rate of the Ca2+-ATPase two phosphoenzymes with that are to PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). we that the interactions with the aspartyl oxygens be weaker for and for and the interactions with the phosphate oxygens be two to the phosphate oxygens and to the aspartyl oxygens in the The is from the bridging in the structure and the phosphate group an with at one of the phosphate oxygens in with in the structure G. PubMed Scopus Google Scholar). the structure of two phosphate oxygens are to two in a and the bridging is which a bond to the with an bond valence of The in The Press, Scholar) the of which is weaker observed with Mg2+ in the other W. A. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar) with bond the of of the with the was in the state with aspartyl phosphate that the phosphoenzyme and is the protein where the interactions with the aspartyl oxygens are from the is to the bridging and Mg2+ Å from the The between and the of the between acetyl phosphate and E2-P to be a for the that the of and E2-P. The aspartyl phosphate to be in a acetyl phosphate in and phosphate are found at A. W. Biophys. PubMed Scopus Google Scholar). we that the relative of noncovalent to the phosphate and aspartyl oxygens is one of the that the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins. to the phosphate oxygens and to the aspartyl oxygens and the bridging P–O which the catalytic of the of the P–O bond is by bond is an of aspartyl phosphate to a shift of interactions from phosphate to aspartyl oxygens. the relative can be by the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens with in This an for the to hydrolysis. We W. for and of W. for the of M. for for for for and for and the bond valence

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