Abstract
Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-A resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.
Highlights
Glycosylasparaginase (GA)1 is an amidase involved in Asnlinked glycoprotein degradation [1]
GA Eluted as a Single Peak from Gel Filtration Column— Previous reports concerning the native GA quaternary structure drew conclusions mainly based on the apparent molecular masses on sizing chromatography or native gel electrophoresis, often using samples from cell extracts and visualizing a trace amount of GA by Western blot methods
Due to the loss of its critical nucleophile for autoproteolysis, we are able to purify a large quantity of precursor protein of the T152A mutant
Summary
Protein Purification—Proteins were expressed and purified by existing procedures [21], with some modifications. Fusion proteins were affinity-purified from the crude extracts over an amylose column according to the protocol of the manufacturer (New England Biolabs). To obtain GA proteins, the purified fusion proteins were further digested with factor Xa at room temperature overnight, and GA was separated from MBP and factor Xa with a HiTrap Q-Sepharose (Amersham Biosciences) column. Gel Filtration—A 200-l volume, 20 M each of the GA or MBP fusion proteins, was applied to an Amersham Biosciences HiPrep 16/60 Sephacryl S-200 HR gel filtration column, equilibrated in a solution containing 10 mM potassium-phosphate buffer, pH 7.4, and 1 mM EDTA. Protein concentrations were 2–5 M in 10 mM potassium-phosphate buffer, pH 7.4, and 1 mM EDTA and were determined by absorbance measurements in 6 M guanidine hydrochloride [22]. ORIGIN software (Microcal, Inc.) was used for the CD analyses and display
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