Organization of the functional domains of anthranilate synthase from Neurospora crassa. Limited proteolysis studies.
Treatment of the multifunctional alpha 2 beta 2 anthranilate synthase complex of Neurospora crassa with elastase produced two fragments of the complex, one possessing anthranilate synthase activity and the other having both indole-3-glycerol phosphate (InGP) synthase and N-(5'-phosphoribosyl)anthranilate (PRA) isomerase activities. Sequencing the NH2 terminus of the InGP synthase-PRA isomerase fragment revealed that cleavage was between positions 237 and 238 of the beta-subunit within a segment of the polypeptide chain which links the glutamine-binding (G) domain with the InGP synthase-PRA isomerase domains. The fragment containing anthranilate synthase activity has a molecular weight of 98,000, as estimated by gel filtration, and is composed of an apparently intact alpha-subunit (70 kDa) associated with the G-domain fragment (29 kDa) derived from the beta-subunit. The alpha X G-domain complex was resistant to further degradation by elastase. When either the alpha 2 beta 2 complex or the alpha X G-domain complex was incubated with trypsin, the alpha-subunit was degraded to a 66-kDa alpha-fragment with reduced enzymatic activity, which was resistant to further cleavage. In contrast, incubation of alpha-subunit alone with either elastase or trypsin resulted in its complete degradation, indicating that association of the alpha-subunit with either G-domain or beta-subunit protected the alpha-subunit from this extensive degradation. A model for the anthranilate synthase complex is proposed in which the trifunctional beta-subunit forms a dimer by the self-association of the InGP synthase-PRA isomerase domains; the G-domain is connected to the InGP synthase-PRA isomerase domain by a relatively disordered region of the peptide chain which, in the alpha 2 beta 2 complex, remains susceptible to proteases; and neither alpha-subunit nor G-domain significantly self-associates.
D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000032:analysis* / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D005973:Glutamine; D010196:Pancreatic Elastase; D014357:Trypsin; D000878:Anthranilate Synthase