PLP cofactor binding

Role of a glycine-rich loop as a PLP cofactor-binding site... 

Kabil O, Toaka S, LoBrutto R, Shoemaker R, and Banerjee R (2001) Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme. Journal of Biological Chemistry 276,19350-5. 

As previously mentioned, tryptophanase is inhibited by a variety of different amino acids, which react with the PLP-cofactor to form covalent intermediates (102), but the structure of the substrate prevents completion of the reaction pathway. At equilibrium, these quasisubstrates generally form intense absorption bands in the 500-nm region of the spectrum, which result from the accumulation of a stable quinonoidal species. Phillips et al. (105) utilized RSSF in conjunction with SWSF studies to investigate the mechanisms of reaction for various aminoacid analogs of i-Trp in order to determine substrate structural elements important both for substrate binding and reactivity with the enzyme (structures 6-9). 

A PLP cofactor attached to the lysine in an His-Lys-X-X-X-Pro-X-Gly-X-Gly motif is a crucial feature of these proteins. Additionally crucial is a conserved cysteinyl residue, which serves as the persulfide site. These proteins belong to fold-type 1 of PLP-dependent enzymes and are homodimers. Each monomer is subdivided into a large domain with one molecule of PLP in aldimine linkage with a Lys residue and a small domain, where the critical cysteinyl residue is located in the middle of a loop. An extended lohe in CDS contains the conserved Cys and constitutes one side of the entrance to the active site. This lohe in CSD may he responsible for the ability of the enzyme to discriminate between selenocysteine and cysteine. NifS binds and transforms the cysteine substrate in a manner usual for PLP-containing enzymes up to the stage of the central quinonoid intermediate. Cysteine desulfuration is initiated by the formation of a Schifif base between cysteine and PLP, followed by the abstraction of sulfur from the substrate and formation of an enzyme-bound cysteine persulfide and alanine via a ketimine intermediate. The cysteine residue acts as a nucleophile and attacks the sulfhydryl... 

ALAS was initially isolated and purified from mammalian sources in the 1970s [11, 12]. The instability of the enzyme, its tendency to form aggregates and the low amount of ALAS in mitochondria were the major factors that affected the development of purification procedures conducive to obtaining protein in sufficient amounts to address questions related to the structure and function of ALAS. Even the use of drugs known to induce hepatic ALAS, such as 2-allyl-2-isopropylacetamide and 3,5-di-carbethoxy-l,4-dihydrocollidine, did not increase substantially the yield of purified ALAS from drug-induced animals [58-61]. In all studies, ALAS has been identified as a homodimer with PLP as an essential cofactor [11-13, 61]. The initial studies on the binding of the PLP cofactor were per-... 

Studies of transaminases in kinetic resolution elucidated several benefits. Compared to asymmetric synthesis the equilibrium favors product formation if pyruvate is used as an amino acceptor. To get enantiopure amines, kinetic resolution is an acceptable choice with yields of 50%. Various (R)- and (S)-selective transaminases are well established nowadays, leading to enantiopure (S)- and (R)-amines, with high ee. Unfortunately product inhibition is one major disadvantage of kinetic resolution. If a critical concentration of product is achieved, the maximum conversion is prevented. Based on kinetic modeling in a previous study from Shin and Kim, the inhibitory effects were based on the strong binding of the product to the PLP cofactor. Consequently the binding of the amino acceptor is hindered, and conversion of the substrate is not possible [72]. 

Natural product molecules are biosynthesized by a sequence of reactions which, with very few exceptions, are catalysed by enzymes. Enzymes are protein molecules which facilitate chemical modification of substrates by virtue of their specific binding properties conferred by the particular combination of functional groups in the constituent amino acids. In many cases, a suitable cofactor, e.g. NAD+, PLP, HSCoA (see below), as well as the substrate, may also be bound to participate in the transformation. Although enzymes catalyse some fairly elaborate and sometimes unexpected changes, it is generally possible to account for the reactions using sound chemical principles and mechanisms. As we explore the pathways to a wide variety of natural products, the reactions will generally be... 

Enzymes are proteins that act as biological catalysts. They facilitate chemical modification of substrate molecules by virtue of their specific binding properties, which arise from particular combinations of functional groups in the constituent amino acids at the so-called active site. In many cases, an essential cofactor, e.g. NAD+, PLP, or TPP, may also be bound to participate in the transformation. The involvement of enzymes in biochemical reactions has been a major theme throughout this book. The ability of enzymes to carry out quite complex chemical reactions, rapidly, at room temperature, and under essentially neutral conditions is viewed with envy by synthetic chemists, who are making rapid progress in harnessing this ability for their own uses. Several enzymes are currently of importance commercially, or for medical use, and... 

The glycine-dependent aldolases contain a cofactor pyridoxal phosphate (PLP). Binding of glycine to it as an imine enables the deprotonation necessary for the carbon-carbon bond forming reaction, with pyridine acting as an electron sink. The subsequent 100% atom efficient reaction with an aldehyde establishes the new bond and two new stereocenters (Scheme 5.30). Of all the glycine-dependent aldolases only L-threonine aldolase (LTA) is commonly used [40, 43, 52]. 

In this chapter the main topic for discussion are the GAIs of GP, so of the above mentioned binding sites, the main concern is the catalytic site. This catalytic site is a deep cavity located at the center of the whole protein, 15 A from the protein surface, and close to the essential cofactor pyridoxal 5 -phosphate (PLP) It has been probed with glucose and GAIs [4]. 

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