A Detailed Enzyme Investigation

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Presentation transcript:

A Detailed Enzyme Investigation Protein Kinase C A Detailed Enzyme Investigation

What’s a Kinase? A kinase is a protein that adds a phophate group from ATP to another protein (phosphorylation). Phosphorylating a protein changes its characteristics and behaviors. This often means that phosphorylating a protein activates it from and inactive to an active state.

Protein Kinase C Comes in a variety of isoforms- 10 in total Generally categorized into two forms Calcium ion dependent (we will study some of these) Calcium ion independent

Four Regions of PKC C1- Binds diacyl glycerol- an activation site C2- Calcium binding site- an activation site C1 and C2 serve to regulate the enzyme- If PKC was not regulated, it could not serve as a “switch” to activate protiens. It would turn every protein it could interact with to the “on” position. C3 and C4 – Form the ATP and substrate binding lobes. Ref 1

Mechanism of Reaction Ref 2 In this reaction, X is a nucleophile, typically an atom with lone pairs such as an oxygen from tryosine. R is the remainder of the protein.

Primary Structure of PKC ADVFPGNDSTASQDVANRFARKGALRQKNVHEVKDHKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKRCHEFVTFSCPGADKGPDTDDPRSKHKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMNVHKQCVINVPSLCGMDHTEKRGRIYLKAEVADEKLHVTVRDAKNLIPMDPNGLSDPYVKLKLIPDPKNESKQKTKTIRSTLNPQWNESFTFKLKPSDKDRRLSVEIWDWDRTTRNDFMGSLSFGVSELMKMPASGWYKLLNQEEGEYYNVPIPEGDEEGNMELRQKFEKAKLGPAGNKVISPSEDRKQPSNNLDRVKLTDFNFLMVLGKGSFGKVMLADRKGTEELYAIKILKKDVVIQDDDVECTMVEKRVLALLDKPPFLTQLHSCFQTVDRLYFVMEYVNGGDLMYHIQQVGKFKEPQAVFYAAEISIGLFFLHKRGIIYRDLKLDNVMLDSEGHIKIADFGMCKEHMMDGVTTRTFCGTPDYIAPEIIAYQPYGKSVDWWAYGVLLYEMLAGQPPFDGEDEDELFQSIMEHNVSYPKSLSKEAVSICKGLMTKHPAKRLGCGPEGERDVREHAFFRRIDWEKLENREIQPPFKPKVCGKGAENFDKFFTRGQPVLTPPDQLVIANIDQSDFEGFSYVNP QFVHPILQSAV

Kylie-Doolittle Hydrophobicity Plot- Regions above zero are hydrophobic Ref 3

Hopp-Woods- Regions above 0 are hydrophillic- Note regions below zero- Possible C1, C2, C3, C4

Secondary Structures in PKC An entire PKC protein has never been analyzed using X-ray crystallography, but portions of the PKC protein have been analyzed. In the secondary protein structures that have been crystalized alpha helices and beta sheets are found. (PDB 1BDY). The next slide shows alpha helices (purple and pink) and beta sheets (green) in a proposed model of the active site.

Ref 4

Tertiary Structure As stated no tertiary structure has been observed for an entire PKC molecule, but a tertiary structure of the has been proposed by Orr and Newtown based on the structure of Protein Kinase A.

Ref 4

With ATP and Sample Sustrate Ref 4

Kinetics of PKC Km = 4.3 mM (Ref 6) Kcat varies depending on the concentration of Ca and diacyl glycerol (Ref 6)

References 1)http://www.promega.com/guides/sigtrans_guide/strg_chap03.pdf 2)Parang, K, Till, J, Ablooglu, A, Kohanski, R, Hubbard, S, Cole, P, Mechanism-based design of a protein kinase inhibitor, Nature Structural Biology, 8(1): 38-41. 3)http://www.vivo.colostate.edu/molkit/hydropathy/index.html 4)http://cti.itc.virginia.edu/~cmg/Demo/pdb/pkc/pkc.html 5) http://www.kinasenet.org/pkr/simplesearch.do - Search- PKC Entry- 1079 6) Hannun, Y, Bell, R, (1990) Rat brain protein kinase C: kinetic analysis of substrate dependence, allosteric regulation, and autophophorylation, Journal of Biological Chemistry, 265(5):2962-2972.