title: Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants.
abstract: Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.
authors.affiliation: Research Reactor Institute, Faculty of Science, Kyoto University, Kumatori, Sennan, Osaka, Japan.
authors.forename: Tooru
authors.initials: T
authors.lastname: Nakamura
authors.forename: Miyo
authors.initials: M
authors.lastname: Sakai
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Sadakane
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Tadatoshi
authors.initials: T
authors.lastname: Kinouchi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Saito
authors.forename: Noriko
authors.initials: N
authors.lastname: Fujii
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D001224:Aspartic Acid; D001216:Asparagine