Mutational analysis of two PWW sequence motifs in human aminoacylase 1.
Human and porcine aminoacylase 1 (Acy1) contain two peculiar sequence motifs located near the interface between the two major domains of the Acy1 subunit. Each motif consists of the sequence PWW preceded by four strongly polar residues. In order to examine the significance of these sequences for Acy1 stability and/or catalysis, we used site-directed mutagenesis of human Acy1 to replace the tryptophan residues in either motif with alanines. Both mutants showed unchanged zinc binding and normal substrate affinity. Modification in PWW motif 1 (residues 192 -194) and motif 2 (residues 321 - 323) resulted in markedly reduced specific activity in the first case and diminished stability in either mutant. Fluorescence quenching measurements showed that all four tryptophans of the PWW motifs are solvent-accessible. We conclude that PWW motif 1 maintains the native conformation of the active site by creating the proper spatial relationship between dimerization domains and catalytic domains, while motif PWW2 is necessary for the stability of the catalytic domain.
Lindner
H
H
Institute of Physiological Chemistry, School of Medicine, Philipps-University, Marburg, Germany.
Berens
W
W
Kraus
I
I
Röhm
K H
KH
D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D004795:Enzyme Stability; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D007454:Iodides; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000166:cytology; D015032:Zinc
D007454:Iodides; D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc