4zm9
From Proteopedia
Crystal structure of circularly permuted human asparaginase-like protein 1
Structural highlights
FunctionASGL1_HUMAN Has both L-asparaginase and beta-aspartyl peptidase activity. May be involved in the production of L-aspartate, which can act as an excitatory neurotransmitter in some brain regions. Is highly active with L-Asp beta-methyl ester. Besides, has catalytic activity toward beta-aspartyl dipeptides and their methyl esters, including beta-L-Asp-L-Phe, beta-L-Asp-L-Phe methyl ester (aspartame), beta-L-Asp-L-Ala, beta-L-Asp-L-Leu and beta-L-Asp-L-Lys. Does not have aspartylglucosaminidase activity and is inactive toward GlcNAc-L-Asn. Likewise, has no activity toward glutamine.[1] Publication Abstract from PubMedThe human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an alphabeta-betaalpha sandwich fold homodimer that cleaves its own peptide backbone at the G167-T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167-T168 peptide bond reaches only </=50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure-activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member. Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages.,Li W, Irani S, Crutchfield A, Hodge K, Matthews W, Patel P, Zhang YJ, Stone E Biochemistry. 2016 Feb 16;55(6):960-9. doi: 10.1021/acs.biochem.5b01157. Epub, 2016 Feb 2. PMID:26780688[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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