Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck delta II crystallin.
Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity. At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate. The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis. On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined. Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity. Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities. During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved [Vallee et al. (1999) Biochemistry 38, 2425-2434], which has allowed the kinetic data to be interpreted on a structural basis.
Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
A R
AR
Chakraborty
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A
Davidson
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Howell
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001123:Argininosuccinate Lyase / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D004372:Ducks; D004789:Enzyme Activation / Q000235:genetics; D019791:Guanidine; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012984:Software; D013379:Substrate Specificity / Q000235:genetics
D000596:Amino Acids; D003459:Crystallins; D001123:Argininosuccinate Lyase; D019791:Guanidine