1994
DOI: 10.1002/pro.5560031121
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Abstract: To probe the role of the Asp-99. . .His-48 pair in phospholipase A, (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99 + Asn-99 (D99N) of bovine pancreatic PLAZ was undertaken. Crystals of D99N belong to the trigonal space group P3121 and were isomorphous to the wild type (WT) (Noel J P et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that … Show more

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Cited by 31 publications
(60 citation statements)
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“…Unlike PI-PLC and serine proteases, the SSHB in PLA 2 exists between the N ␦1 of His 48 and one of the nonbridging phosphonate oxygen atoms in the transition state analog, whereas a normal H-bond is formed between the N ⑀2 of His 48 and the O ␦1 of Asp 99 at low pH or in the presence of the transition state analog (HK32). Similar to that has been observed for the mutant D274N PI-PLC, both the x-ray crystal structure and NMR experimental results suggest that a regular HB exists between Asn 99 and protonated His 48 in the D99N mutant of sPLA 2 , and D99N still preserves 5% of the catalytic activity compared with that of WT (27,28).…”
Section: Dissecting the Roles Of The H-bond And The Negative Charge Osupporting
confidence: 80%
“…Unlike PI-PLC and serine proteases, the SSHB in PLA 2 exists between the N ␦1 of His 48 and one of the nonbridging phosphonate oxygen atoms in the transition state analog, whereas a normal H-bond is formed between the N ⑀2 of His 48 and the O ␦1 of Asp 99 at low pH or in the presence of the transition state analog (HK32). Similar to that has been observed for the mutant D274N PI-PLC, both the x-ray crystal structure and NMR experimental results suggest that a regular HB exists between Asn 99 and protonated His 48 in the D99N mutant of sPLA 2 , and D99N still preserves 5% of the catalytic activity compared with that of WT (27,28).…”
Section: Dissecting the Roles Of The H-bond And The Negative Charge Osupporting
confidence: 80%
“…The kinetic properties of the D99N mutant constructed previously were also further analyzed. The X-ray structure of the Y52,73F/D99N mutant indicated a substantial disruption of the hydrogen-bonding network including the loss of the structural water similar to that seen in the structure of the D99N mutant published previously [Kumar, A., Sekharudu, Y. C., Ramakrishnan, B., Dupureur, C. M., Zhu, H., Tsai, M.-D., & Sundaralingam, M. (1994) Protein Sci. 3, 2082-2088.…”
supporting
confidence: 74%
“…The crystal structure of the double mutant Y52,73F (Sekharudu et al, 1992) showed that although the loss of hydrogen bonds of the tyrosines was compensated by the increase in the hydrophobic contacts of the phenyl groups, the structural water was retained. However, in the single mutant D99N (Kumar et al, 1994), the structural water was absent.To gain a better understanding of the role of the conserved structural water in hydrogen bonding to the catalytic triad and the active site residues, we have undertaken detailed analyses of the triple mutant Y52,73F/D99N of bovine pancreatic PLA2. The crystal structures of the triple mutant were solved in both trigonal and orthorhombic forms at 1.9 and 1.8 Å, respectively.…”
mentioning
confidence: 97%
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“…By changing Asp260 into asparagine, some of the hydrogen bonds would surely be lost or weakened, thereby disrupting or destabilizing the hydrogen-bonding networks. This may lead to conformational perturbations as was observed in phospholipase A 2 (Li & Tsai, 1993;Kumar et al, 1994). The contribution of buried hydrogen-bonding networks to protein stability was also demonstrated in acetylcholinesterase (Ordentlich et al, 1993), barnase (Chen et al, 1993), and lactate dehydrogenase (Nobbs et al, 1994).…”
Section: Discussionmentioning
confidence: 78%