Cysteine 5 -phosphate

In the biosynthesis of the thia2ole, cysteine is the common sulfur donor. In yeasts, the C-2 and N may be suppHed by glycine, and the remaining carbons byD-ribulose-5-phosphate [108321-99-9] (50). In anaerobic bacteria, the C-2 andN maybe recmited from tyrosine and the carbons from D-l-deoxyxylulose [16709-34-5] (51), whereas in aerobic bacteria the C-2 and N maybe derived from glycine, as in yeasts 7 (74—76,83—86) (see Fig. 9). 

FIGURE 16.10 Formation of a covalent intermediate in the glyceraldehyde-3-phos-phate dehydrogenase reaction. Nucleophilic attack by a cysteine —SH group forms a covalent acylcysteine intermediate. Following hydride transfer to NAD, nucleophilic attack by phosphate yields the product, 1,3-bisphosphoglycerate. 

The amino acid methionine is biosynthesized by a multistep roule that includes reaction of an inline of pyridoxal phosphate (PLP) to give an unsaturated imine. which then reacts with cysteine. What kinds of reactions are occurring in the two steps ... 

The reaction occurs by two sequential nucleophilic acyl substitutions, the first by a cysteine residue in the enzyme, with phosphate as leaving group, and the second by hydride donation fromNADH, with the cysteine residue as leaving group. 

In a series of papers, Cook et al.60-63 presented results of the 31P NMR studies of pyridoxal 5 -phosphate dependent enzyme. O-acetylserine sulf-hydrylase is the enzyme which catalyses the final step of biosynthesis of l-cysteine, the replacement of p-acetoxy group of O-acetyl-L-serine by thiol [30] in bacteria and plants. 

Penicillamine reacts with pyridoxal-5-phosphate to form a thiazolidine derivative, and is able to displace many amino acids from their Schiff base complexes, forming stable compounds of this type. The reactivity of the thiol group of penicillamine is less than that of cysteine, probably because of steric hindrance by the adjacent methyl groups of penicillamine, which in consequence is less rapidly oxidized in vivo [7]. 

Prepare a set of standards by dissolving cysteine in 0.1M sodium phosphate, pH 8.0, at an initial concentration of 2mM (3.5mg/ml) and serially diluting this solution (1 1) with reaction buffer down to at least 0.125 mM. This will produce five solutions of cysteine for generating a standard curve. If a more dilute concentration range is required, continue to serially dilute until a set of standards in the desired range is obtained. 

Wash 0.5 ml of immobilized papain (Thermo Fisher) with 4 X 2 ml of 20 mM sodium phosphate, 20mM cysteine-HCl, 10 mM EDTA, pH 6.2 (digestion buffer), and finally suspend the gel in 1.0 ml of digestion buffer. 

Alkaline phosphatases [AP, orthophosphoric-monoester phosphorylase (alkaline optimum) EC 3.1.3.1] represent a large family of almost ubiquitous isoenzymes found in organisms from bacteria to animals. In mammals, there are two forms of AP, one form present in a variety of tissues and another form found only in the intestines. They share common attributes in that the phosphatase activity is optimal at pH 8-10, is activated by the presence of divalent cations, and is inhibited by cysteine, cyanides, arsenate, various metal chelators, and phosphate ions. Most conjugates created with AP utilize the form isolated from calf intestine. 

FIGURE 6.1 CVs obtained at cysteine-modified (curves a and b) and bare Au (curve c) electrodes in 25 mM phosphate buffer in the presence (curves a and c) and absence (curve b) of 0.56 mM Cu, Zn-SOD. Potential scan rate, lOOmV s-1. (Reprinted from [98], with permission from Elsevier.)... 

Similar to those observed with the cysteine-modified electrode in Cu, Zn-SOD solution [98], CVs obtained at the MPA-modified Au electrode in phosphate buffer containing Fe-SOD or Mn-SOD at different potential scan rates (v) clearly show that the peak currents obtained for each SOD are linear with v (not v 1/2) over the potential scan range from 10 to 1000 mVs-1. This observation reveals that the electron transfer of the SODs is a surface-confined process and not a diffusion-controlled one. The previously observed cysteine-promoted surface-confined electron transfer process of Cu, Zn-SOD has been primarily elucidated based on the formation of a cysteine-bridged SOD-electrode complex oriented at an electrode-solution interface, which is expected to sufficiently facilitate a direct electron transfer between the metal active site in SOD and Au electrodes. Such a model appears to be also suitable for the SODs (i.e. Cu, Zn-SOD, Fe-SOD, and Mn-SOD) with MPA promoter. The so-called... 

FIGURE 6.8 (a) Typical current-time response obtained at the Cu, Zn-SOD/cysteine-modified Au electrode in phosphate buffer (02-saturated) solution containing 0.002 unit of XOD upon the addition of 50 nM xanthine and the subsequent addition of 6 j,M Cu, Zn-SOD. (b) Current-time responses of (i) the apo-SOD/cysteine-modified Au electrode and (ii) the Cu, Zn-SOD/cysteine-modified Au electrode toward 02 in phosphate buffer containing 0.002 unit of XOD upon the addition of 40 nM xanthine. The electrode was polarized at +300mV, and the solution was gently stirred with a magnetic stirrer at 200rpm. (Reprinted from [151], with permission from the American Chemical Society.)... 

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