Phospholipase design

The breakdown of glycerophospholipids is catalysed by a series of phospholipases designated A, B, C and D depending on their positions of attack (Fig. 11.16). In addition, phosphatidic acid phosphatase is important, but this has already been dealt with (Section 11.2.1). Two phospholipase A specificities are recognized and these are named Ai and A2 depending on the position of the ester hydrolysed in the diacylphosphoglyceride. Phospholipase B is the name usually used for an enzyme which is a... 

Serotoninergic System. Figure 1 Graphical representation of the current classification of 5-hydroxytryptamine (5-HT) receptors. Receptor subtypes represented by shaded boxes and lowercase designate receptors that have not been demonstrated to definitively function in native systems. Abbreviations 3-5r cyclic adenosine monophosphate (cAMP) phospholipase C (PLC) negative (-ve) positive (+ve)... 

Like proteases, phospholipases (PLs) are enzymes that catalyze the hydrolysis of a covalent bond, in this particular case of phospholipids. For this reason, a similar approach for the design of FRET... 

The G-protein that has been termed Gp, and that is linked to phospholipase C activation, may in fact be Gaj 2 or Gc. 3. Ga is designated as the G-protein responsible for activation of phospholipase A2, which results in arachidonic acid release. Some experimental evidence indicates that, at least in HL-60 cells, different agonists can preferentially activate different phospholipases, and some of these are responsible for the activation of secretion. In neutrophils, the two pertussis-toxin-sensitive Ga-proteins (Gaj-2 and G j 3) have been identified by peptide mapping of proteolytic digests of the proteins, by peptide sequencing and by immunoblotting. Complementary-DNA clones for the mRNA of these two molecules have also been isolated from an HL-60 cDNA library. Gai-2 is five to ten times more abundant than Gai.3, the former component comprising 3% of the total plasma membrane proteins. It is possible that these two different Ga-subunits are coupled to different phospholipases (e.g. phospholipases C and D). Pertussis toxin inhibits the secretion of O2 after stimulation of neutrophils by fMet-Leu-Phe, but pertussis-toxin-insensitive G-proteins are also present in neutrophils. These may be members of the Gq family and may be involved in the activation of phospholipase Cp (see 6.3.1). 

MICELLAR SUBSTRATES. Phospholipids in micelles are frequently found to be more active substrates in lipolysis than those phospholipids residing in a lipid bilayer". Dennis first described the use of Triton X-100 to manipulate the amount of phospholipid per unit surface area of a micelle in a systematic analysis of the interfacial interactions of lipases with lipid micelles. Verger and Jain et al have presented cogent accounts of the kinetics of interfacial catalysis by phospholipases. The complexity of the problem is illustrated in the diagram shown in Fig. 2 showing how the enzyme in the aqueous phase can bind to the interface (designated by the asterisk) and then become activated. Once this is achieved, E catalyzes conversion of S to release P. ... 

Simpson LL (2000) Identification of the characteristics that underlie botulinum toxin potency implications for designing novel drugs. Biochimie 82 943-53 Simpson LL, Lautenslager GT, Kaiser II, Middlebrook JL (1993) Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects. Toxicon 31 13-26... 

Design and Development of a Selective Assay System for the Phospholipase A2 Superfamily... 

In mammalian cells, there are multiple forms of enzymes within the same family, e.g., protein kinase C, phospholipase C, and caspase. A selective assay system would be instrumental in such cases to advance understanding of the respective role for each form within the same enzyme family. An example for design of a selective assay system for the superfamily of phospholipase A2 is provided in this chapter. Such a selective assay system may play a signihcant enabling role for PLA2 and other enzyme families in the discovery of inhibitors relevant to the treatment of pathologies involving those enzymes. 

RW Schevitz, NJ Bach, DG Carlson, NY Chirgadze, DK Clawson, RD Dillard, SE Draheim, LW Hartley, ND Jones, ED Mihelich, JL Olkowski, DW Snyder, C Sommers, JP Wery. Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2. Nat Struct Biol 2 458-465,1995. 

In a study designed to investigate the structural features of a phospho-glyceride interaction with a bacterial phospholipase C, El-Sayed et al. (1985), reported that the carbonyl group and its closely related environment are most important. A more detailed treatment of the substrate specificity of this enzyme can be found in an excellent review by Massing and Eibl (1994). 

The examples provided in this Chapter demonstrate that directed evolution resembles a very useful tool to create enzyme activities hardly accessible by means of rational protein design (Table 14.1). Even if the desired substrate specificity is known from other biocatalysts - e.g. phospholipase A1 activity - the advantage of the directed evolution approach resides in the already achieved functional expression of a particular protein. Thus bottlenecks arising from the identification of enzymes by traditional screening and cultivation methods can be circumvented. In addition, directed evolution can dramatically reduce the time required for the provision of a suitable tailor-made enzyme, also because cloning and functional expression of the biocatalyst has already been achieved. 

Kauffmann, I., Schmidt-Dannert, C. (2001), Conversion of Bacillus stearother-mophilus lipase into an efficient phospholipase with increased activity towards long chain fatty acyl substrates by directed evolution and rational design, Protein Eng., in press. 

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