Ultra-pure ammonium sulfate was from ICN Biomedicals, Inc
Ultra-pure ammonium sulfate was from ICN Biomedicals, Inc. stronger than predicted for independently acting inhibitors. This indicates that in some cases inhibition of homodimeric enzymes by mixtures of inhibitors (heteroinhibition) may offer advantages over single inhibitors. aspartate transcarbamoylase . More recently discovered is flip-flop catalysis, postulated in thiamin pyrophosphate-dependent enzymes. In this family of homodimeric enzymes two subunits alternatively catalyze half-reactions coupled so that while the first half-reaction proceeds in one subunit the second half-reaction takes place in the other subunit . Yet another concept is that the subunits of oligomeric enzymes communicate so the energy of substrate binding in one Sulbutiamine subunit is transferred to the other and used to release product [6,7]. This feature should Sulbutiamine be most valuable for enzymes such as enolase that rapidly catalyze reactions in both the forward and reverse directions since such enzymes must strongly bind product. Dimers interacting in such a way must be asymmetric when substrate/product molecules are bound to Sulbutiamine them. They are likely to show negative cooperativity between Sulbutiamine subunits for binding of substrates or inhibitory substrate analogues. The mechanism of enolase is shown in Figure 1. It was originally proposed by Reed and coworkers and modified to include the role of His157; it is based on results from two laboratories [8,9]. The three crucial residues all function as acid/base systems. In the glycolytic direction of the enolase reaction, Lys345 abstracts a proton from C2 of PGA, Glu209, likely augmented by Glu166 and His370, protonates the hydroxyl at C3, while His157 protonates the phosphate moiety to produce electron withdrawing from C2 [9,10]. In the gluconeogenetic direction of catalysis, the roles of Lys342 and Glu209 are reversed while His157 keeps away from the phosphate. The catalytic loops positions change upon PGA binding from Rabbit Polyclonal to PKA-R2beta (phospho-Ser113) the open conformation to the closed conformation. In the complex with PEP, loop 155C159 is in a third conformation, referred to as semi-closed in which His157 does not directly contact the phosphate moiety but through a water molecule . Enolase catalysis is also at least in part entatic as the PGA when bound to the enzyme is in a conformation which is different from that observed in the unbound state and which is likely of a higher energy . Open in a separate window Figure 1 Mechanism of enolase. The hNSE amino acid numbering is used. We use Glu209 for the Glu166, Glu209, His370 cluster surrounding the hydroxyl. In yeast enolase 1, His 157 is His159, Glu209 is Glu211, and Lys342 is Lys 345. The carboxylate carbon is C1, the phosphorylated carbon is C2 and the last one is C3.t In vertebrate organisms three isozymes of enolase, expressed by different genes, are present. Enolase is ubiquitous; enolase is muscle specific, and enolase (NSE) is neuron specific. Sequence comparisons show 83% identity and 87% similarity for each pairing of the isozymes of human enolase. Tissue specific isozymes ( and ) readily form mixed dimers with enolase . Human NSE (hNSE) is a major brain protein that constitutes between 0.4 to 2.2% of the total soluble protein of brain, depending on region. In some neurons NSE accounts for 3C4% of total soluble protein, which led to common usage of NSE as a clinical marker for neuronal and neuroendocrine cells . The reasons for such a high concentration of enolase in neurons are not known, though neurons depend entirely on glycolysis as the source of acetyl SCOA and hence energy . In muscle cells, where the concentrations of all glycolytic enzymes are high, enolase is about.