BCH Graduate Survey of Biochemistry

Transcription

1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Marsha Durosier Lecture 9 Slide sets available at:

2 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 12 Biosignaling 2008 W. H. Freeman and Company

3 Nitric oxide (NO) is a gaseous signaling molecule and neurotransmitter. Its role in biology was established in 1987 by Palmer et al. NO acts as an endothelium-derived relaxing factor that mediates the relaxation of arterial wall smooth muscle (SM) causing vasodilation. The EDRF (NO) is released in response to mechanical force and neurohumoral mediators acetylcholine, bradykinin and histamine. NO stimulates muscle guanylate cyclase activity leading to an increase in cgmp and muscle relaxation. cgmp-induced SM relaxation is mediated mainly by cgmp-dependent protein kinase activation. Interestingly, free radicals of NO act as a neurotransmitter in nonadrenergic noncholinergic nerves in the peripheral nervous system. NO exists in several oxidation-reduction states: a reduced form NO, correctly termed nitric oxide; NO +, a nitrosonium ion; and finally NO or nitric monoxide.

4 NO signaling involves several molecular events culminating in a reduction in intracellular Ca 2+ concentration and a decrease in the sensitivity of the contractile system to Ca 2+. NO is also a neurotransmitter involved in intestinal relaxation in peristalsis and is also important in penile erection. It is considered to be involved in various effects of the neurotransmitter glutamate acting at NMDA receptors in the brain. NO is synthesized from L-arginine in neurons and other cells by the enzyme nitric oxide synthase.

5 Palmer RM, Ferrige AG, Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: Endothelium-derived relaxing factor (EDRF) is a labile humoral agent, which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. NO was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentrationdependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.

6 Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, Osterloh IH, Gingell C (1996) Sildenafil: an orally active type 5 cyclic GMPspecific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 8: Sildenafil (Viagra, UK-92,480) is a novel oral agent under development for the treatment of penile erectile dysfunction. Erection is dependent on nitric oxide and its second messenger, cyclic guanosine monophosphate (cgmp). However, the relative importance of phosphodiesterase (PDE) isozymes is not clear. We have identified both cgmp- and cyclic adenosine monophosphate-specific phosphodiesterases (PDEs) in human corpora cavernosa in vitro. The main PDE activity in this tissue was due to PDE5, with PDE2 and 3 also identified. Sildenafil is a selective inhibitor of PDE5 with a mean IC50 of 3.9 nm. In human volunteers, we have shown sildenafil to have suitable pharmacokinetic and pharmacodynamic properties (rapid absorption, relatively short half-life, no significant effect on heart rate and blood pressure) for an oral agent to be taken, as required, prior to sexual activity. Moreover, in a clinical study of 12 patients with erectile dysfunction without an established organic cause, we have shown sildenafil to enhance the erectile response (duration and rigidity of erection) to visual sexual stimulation, thus highlighting the important role of PDE5 in human penile erection. Sildenafil holds promise as a new effective oral treatment for penile erectile dysfunction.

7 Because the NO-cGMP signaling pathway is a component of several biological processes, compounds that specifically influence the pathway have potentially important therapeutic uses and significant economic value. I am sure most everyone will recognize the popular name of at least one such compound. So biochemically what exactly does sildenafil do? 7CFD454D98ABD&first=0&FORM=LKVR2 Can you figure out how to make NO from L-arginine? Viagra is among the company s top 10 best-selling drugs, behind such products as Lipitor, Zoloft and Celebrex. Viagra accounted for $1.87 billion in global sales last year, including $1.1 billion in sales to American men. Excerpt from: MSNBC Pfizer ordered to pull Viagra commercials Associated Press; Updated: 9:22 a.m. ET Nov 16, 2004

8 Image provided by H. Criss Hartzell with permission. From H. Criss Hartzell, The Stress of Relaxation Science 317: September 2007

9 Figure 1. NO-cGMP signaling pathway. From : Bivalacqua TJ, Champion HC, Hellstrom WJ, Kadowitz PJ (2000) Trends Pharmacol Sci 21:

10 This slide is not in the lecture video NO chemistry. NO is a free radical and while free radicals are, by definition, generally unstable and highly reactive, however NO is rather chemically stable. NO does react readily with other free radicals such as O 2 (superoxide) or lipid peroxyl radicals. NO does form stable high affinity bonds with ferrous hemes and nonheme iron which is necessary for the activation of the guanylate cyclase activity. Overall, NO is uncharged and is surprisingly perhaps, highly soluble in hydrophobic environments and this allows for its free diffusion across biological membranes. Its small size and solubility in nonpolar environments allows it to serve as a rapidly diffusible signal distant from its site of synthesis. The half-life for NO in some tissues is estimated to be in the range of s to longer than 2 s.

11 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 4 The Three-Dimensional Structure of Proteins 2008 W. H. Freeman and Company

12 This is a depiction of folding from a random chain to secondary and tertiary structure

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16 In 1951, Pauling and Corey published seven papers back-to-back in PNAS on the atomic coordinates for two helical configurations of polypeptides, the pleated sheet structure, the structure of keratin in a feather rachis, the structure of hair and muscle and collagen fibers and the polypeptide configurations in hemoglobin and other globular proteins that would rock the biochemistry world. And, without question their work has provided an important key to understanding the functions of proteins at the molecular level. Herman Branson was an early collaborator with Pauling and Corey and helped them to decipher and verify protein structures. Pauling and Corey were an original odd couple, or a perfect case where opposites attract, a paradigm well known in biochemistry. Pauling was a showman and loved to entertain and charm scientific audiences, but Corey disliked social events and did not like to attract attention to himself. Their collaboration would last almost three decades. Pauling and Corey used x-ray crystallography of short peptides from several proteins to elucidate the alpha helix and beta sheet structures of proteins. Pauling would receive the 1954 Nobel Prize in Chemistry for his work on protein structure.

17 Note intrahelical hydrogen bonding The alpha helix shown in ball and stick form and space filling models. If the space fill model (d) were to be rotated to an end on view so that we could look right down the center of the helical axis like in (c) we would see no open space. All the space would take up by the van der Waals radii of the atom electron clouds. What exactly does the formation of an alpha helix represent in terms of the energy status of the helical structure? Does it represent an energy maximum or energy minimum? Hint, what does structural stability reflect?

18 The rotational direction of a helix can be determined you the use of one s hands. If going from N-terminus to C- terminus starting with N- terminus pointing down, if the helix pattern follows your right hand, then it is a right-handed helix. Similarly if the rotational direction follows that of your left hand, then the helix is a left-handed helix. Because the amino-terminus carries a positive charge and the carboxyl-terminus has a negative charge and there is a retention of this orientation for the partial positive and negatives charges of the hydrogen bonds that form along the helical chain, it is state that the alpha helix is a macro-dipole.

19 Note the inter chain hydrogen bonding. Beta pleated sheets occur in two forms, antiparallel and parallel referring to the direction the polypeptide chains are oriented. Either way, the arrangement of a the amino and carboxyl groups are spaced in a way that allows for the formation of the interchain hydrogen bonding. Can this be helpful for protein structure stability? If so, then how? Clearly the peptide bonds in the beta-structure have different φ and ψ bond angles than the alpha helix.

20 Approximately one-third of amino acids in a globular protein can be associated with loops and turns. A common type of turn in proteins is the beta turn that links two regions of beta structure. The turn is usually four amino acids in length and the orientation of the residues are such that a hydrogen bond can form between and first and four amino acids. Proline is often a constituent of beta turns and can occur in the cis instead of the trans conformation typical of virtually all peptide bonds.

21 Ramachandran plots show the range of φ and ψ bond angles in proteins. A survey of many proteins whose 3-dimensional structures are known, shows that the φ and ψ bond angles fall into discrete regions of the bond angle universe. This should not be surprising as different structural forms depend on the arrangement of atoms around the peptide bonds. Then it follows that the different amino acids will favor or disfavor certain types of structures.

22 Hair is composed of alpha Keratin a right-handed alpha helix. See how the organization of the chains, protofilament and protofibril contribute to the structure of the hair shaft. Although it may not seem like it, but this arrangement of keratin chains is designed for strength. Note that the protofilaments overlap in the protofibril. In building construction, we employ the same principles to create strength and stability. The organization of the hair shaft is an example of the quaternary structural organization of proteins.