PubMed · 8900273
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L Lin, S P Hale, P Schimmel. 1996-11-07. Aminoacylation error correction.. https://doi.org/10.1038/384033b0
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The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence energy transfer which yields k(2,f), the rate constant for the fluorescently detected decay of the Michaelis-type complex (Mellet, P., Boudier, C., Mély, Y., and Bieth, J. G. (1998) J. Biol. Chem. 273, 9119-9123); (ii) kinetics of elastase-catalyzed hydrolysis of a substrate in the presence of alpha(1)PI, which yields k(2,e), the rate constant for the conversion of the Michaelis-type complex into irreversibly inhibited elastase. Both rate constants were found to be pH-independent and close to each other, indicating that acylation, a pH-dependent phenomenon, does not govern the decay of the Michaelis-type complex and, therefore, follows translocation. On the other hand, anhydro-elastase reacts with alpha(1)PI to form a Michaelis-type complex that translocates into a second complex with a rate constant close to that measured with active elastase, confirming that acylation is not a prerequisite for translocation. Moreover, the anhydro-elastase-alpha(1)PI complex was found to be thermodynamically reversible, suggesting that translocation of active elastase might also be reversible. We propose that serpins form a Michaelis-type complex EI(M), which reversibly translocates into EI(tr) whose acylation yields the irreversible complex EI(ac). [see text]
The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.
The lipase- and esterase-catalyzed acylations of hydroxylamine and hydrazine derivatives with octanoic acid and ethyl octanoate are described. The influence of solvent and nucleophile on the initial reaction rate was investigated for a number of free and immobilized enzymes. Initial rates were highest in water, but the overall productivity was optimal in dioxane. Octanoic acid (250 g/L) was converted for 93% into the hydroxamic acid in 36 h with only 1% (w/w) Candida antarctica lipase B (Novozym 435) in dioxane at 40 degrees C. This translates to a catalyst productivity of 68.5 g. g(-1). day(-1) and a space time yield of 149 g. L(-1). day(-1), unprecedented figures in the direct reaction of an acid with a nitrogen nucleophile in an organic solvent.