My five eurocents to the discussion ... AMPPNP can also be specifically hydrolyzed by Serca1a, the Sarcoplasmic Reticulum Ca2+-ATPase. Such cleavage was proven to be calcium-dependent meaning that AMPPNP is used by the ATPase as a genuine substrate although with a very slow kinetics compared to what is obtained with the cognate substrate ATP.
J Biol Chem. 1981 Oct 10;256(19):9793-5. Sarcoplasmic reticulum ATPase catalyzes hydrolysis of adenyl-5'-yl imidodiphosphate. Taylor JS. Abstract Sarcoplasmic reticulum ATPase has been found to cleave the ATP analog adenyl-5'-yl imidodiphosphate in a calcium-dependent reaction. The reaction products were determined by 31P NMR to be inorganic phosphate and adenyl-5'-yl phosphoramidate (AMP-PN). AMP-PNP hydrolysis, like ATP hydrolysis, drives active Ca2+ accumulation by sarcoplasmic reticulum vesicles. PMID: 6456267 [PubMed - indexed for MEDLINE] This property allowed us to indeed trap and cystallize the protein under an AMPPN-bound, phosphoenzyme intermediate, that was proposed to be a fair mimick of the so-called E1P (Ca2+).ADP state. Display Settings:AbstractSend to: Nature. 2007 Dec 13;450(7172):1036-42. The structural basis of calcium transport by the calcium pump. Olesen C, Picard M, Winther AM, Gyrup C, Morth JP, Oxvig C, Møller JV, Nissen P. Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Aarhus, Ole Worms Alle, blg. 1185, DK - 8000 Aarhus C, Denmark. Comment in: Nature. 2007 Dec 13;450(7172):957-9. Abstract The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells. PMID: 18075584 [PubMed - indexed for MEDLINE] Martin Picard Le 14 févr. 2011 à 22:53, "Prince, D Bryan" <[email protected]> a écrit : > Hello Steve, > > > > You can also check out this paper: Bystrom, Pettigrew, Remington and > Branchaud (1997) Bioorganic & Medicinal Chemistry Letters, Vol 7 No 20 > pp2613-2616. It describes the creation of AMPPCF2P, which I had opportunity > to use a few years back and it worked great! > > > > Good luck, > > Bryan > > > > > Confidentiality Notice: This message is private and may contain confidential > and proprietary information. If you have received this message in error, > please notify us and remove it from your system and note that you must not > copy, distribute or take any action in reliance on it. Any unauthorized use > or disclosure of the contents of this message is not permitted and may be > unlawful. > > > > > > > > > > From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Derek > Logan > Sent: Monday, February 14, 2011 4:16 PM > To: [email protected] > Subject: Re: [ccp4bb] AMP-PNP Hydrolysis > > > > Hi Steve, > > > > Funnily enough I just read the following paper today, which describes exactly > this phenomenon: > > > > http://www.ncbi.nlm.nih.gov/pubmed/21093442 > > > > Is AMPPCP as sensitive to acid conditions? I would suspect not. > > > > Best wishes > > Derek > > ___________________________________________________________________ > Derek Logan tel: +46 46 222 1443 > Associate Professor fax: +46 46 222 4692 > Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 > Centre for Molecular Protein Science www.cmps.lu.se > Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com > > > > On Feb 14, 2011, at 15:05, Young-Jin Cho wrote: > > > > > Hi Steve, > > With my experience, it is (very) common to see AMPPNP is hydrolyzed to AMPPN > (supposedly) with my protein. Although the literature often reported AMPPNP > as a stable ATP mimic, such a luck wasn't true with my case, maybe same as > you. If you go to Sigma website where I purchased, it may say it is not > stable in an acidic condition. My mother liquor was in an acidic condition. > So you'd better consider if you used it in an acidic condition, otherwise, > your protein inherently has a strong power to hydrolyze it. In addition to > the pH, I often see it can go hydrolysis easily. However, you can try more > as you mentioned it may contain impurity. I just want to inform you that it > is not surprising to see this hydrolysis. > > Good luck~ > > Young-Jin > > > On Mon, Feb 14, 2011 at 8:30 AM, Soisson, Stephen M > <[email protected]> wrote: > > Hi there, > > Was recently looking at a structure of an enzyme with AMP-PNP added to the > crystallization mix, and all I see is density for ADP. I was wondering if > hydrolysis of AMP-PNP to ADP is relatively common - either as a result of > extended time in crystallization or exposure of the resultant crystals to > synchrotron radiation? > > I know that there can be up to 10% contamination of ADP in the purchased > material, so it could just be that we have selected that form in the crystal, > or that there was endogenous ADP bound that failed to substitute. Just > curious if hydrolysis is a common observation. > > Thanks in advance- > > Steve > > Stephen M. Soisson, Ph.D. > Structural Chemistry Site Lead, WP > > Merck Research Laboratories > 770 Sumneytown Pike, WP14-1101 > West Point, PA 19486 > Phone: (215) 652-6185 > Fax: (215) 652-9051 > [email protected] > > > > Notice: This e-mail message, together with any attachments, contains > information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, > New Jersey, USA 08889), and/or its affiliates Direct contact information > for affiliates is available at > http://www.merck.com/contact/contacts.html) that may be confidential, > proprietary copyrighted and/or legally privileged. It is intended solely > for the use of the individual or entity named on this message. If you are > not the intended recipient, and have received this message in error, > please notify us immediately by reply e-mail and then delete it from > your system. > > > >
