Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,

Abstract

The major soluble protein component of avian and reptilian eye lenses, delta crystallin, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses there are two highly homologous delta crystallins, termed delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I crystallin is inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H91N) has been determined to 2.5 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft between three different monomers of the tetrameric protein. A reexamination of the kinetic properties of the H91N mutant reveals that the mutant has 10% wild-type activity. The Vmax of the mutant protein is identical to that of the wild-type protein, but a 10-fold increase in the Michaelis constant is seen, suggesting that His 91 is involved in binding the substrate. In an effort to determine the reasons for the loss of enzymatic activity in delta I crystallin, a structural comparison of the H91N mutant with the enzymatically inactive turkey delta I crystallin has been performed. This study revealed a remarkable similarity in the overall structures of the two proteins. Three regions of secondary structure do differ significantly between the two models; these include the N-terminal tail, a loop containing residues 76-91, and a cis versus trans peptide linkage at residue Thr 322. The cis to trans peptide variation appears to be an interspecies difference between turkey and duck and is therefore not directly involved in the loss of enzymatic activity. All the residues implicated in the catalytic mechanism are conserved in both the active and inactive proteins, and given the linearity of the relationship between the enzymatic activity of duck delta I/delta II heterotetramers and their delta II content (Piatigorsky & Horwitz, 1996), it is evident from the structure that only one of the three domains that contributes to the active site is responsible for the loss of activity in the delta I protein. Given the structural differences found in domain 1 (N-terminal tail and 76-91 loop), we postulate that these differences are responsible for the loss of catalytic activity in the delta I crystallin protein and that the delta I protein is inactive because it no longer binds the substrate.

Mutational Analysis of Duck δ2 Crystallin and the Structure of an Inactive Mutant with Bound Substrate Provide Insight into the Enzymatic Mechanism of Argininosuccinate Lyase

“…Crystal Structure of S283A d␦c2 Mutant with Bound Argininosuccinate-S283A d␦c2 has the same overall architecture as that described previously for other ␦ crystallin (15,17,19,26) and human ASL (18, 42) structures (Fig. 2).…”

“…4 and 6A). Although the roles of Tyr-323, Ser-114, Arg-115, Asn-116, and His-91 have been investigated previously (17,27), no information was available on the role residues Ser-29, Asp-33, Asp-89, Arg-238, Thr-281, Asn-291, Asp-293, Lys-325, Asp-330, and Lys-331 play in substrate binding. The current mutagenesis study found that most of the mutations of putative substrate binding residues had a significant impact on the enzymatic activity.…”

“…Although the overall amino acid sequence homology between these proteins is low (20 -30%), there are three highly conserved regions that are remote from each other in the monomer structure but which cluster together in the tetramer to form the four active sites (15-18). The location of the active site has been confirmed with the structure determination of various superfamily members with bound inhibitors, substrate analogues, or substrates (19 -22).Structural and biochemical studies (23, 24), as well as sitedirected mutagenesis data (25), have suggested that directly (15,17) or via a water molecule (21), is responsible for proton abstraction (base catalysis), whereas Lys-289 has been proposed to stabilize the carbanion intermediate (15,16). Lys-289 belongs to the flexible 280's loop (residues 270 -290), and, although this lysine's role in catalysis is supported by its strict conservation across superfamily, the same is not true for His-162.…”

“…Structural and biochemical studies (23, 24), as well as sitedirected mutagenesis data (25), have suggested that directly (15,17) or via a water molecule (21), is responsible for proton abstraction (base catalysis), whereas Lys-289 has been proposed to stabilize the carbanion intermediate (15,16). Lys-289 belongs to the flexible 280's loop (residues 270 -290), and, although this lysine's role in catalysis is supported by its strict conservation across superfamily, the same is not true for His-162.…”

“…Members of the superfamily include class II fumarase (11), aspartase (12), adenylosuccinate lyase (13), and 3-carboxy-cis,cis-muconate lactonizing (CMLE) enzyme (14). Although the overall amino acid sequence homology between these proteins is low (20 -30%), there are three highly conserved regions that are remote from each other in the monomer structure but which cluster together in the tetramer to form the four active sites (15)(16)(17)(18). The location of the active site has been confirmed with the structure determination of various superfamily members with bound inhibitors, substrate analogues, or substrates (19 -22).…”