Abstract
On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.
Highlights
Iolytic activity at pH 6.2 and a lower binding constantWe converted Trp" of hen egg white lysozyme to Tyr by siteschTftohioamyrindrssillcmtaaiahttruntiltttpteoaooftneorsfttriifhotehiocsoceehsntne.6iote2ooTng.tfhgrTtihehowhuesefhmeleu.iratnHoeenzrpoyleyllwmayssccsoeaeoevztnmzyeicycrmaeem,ncettteshiw,pveoweieftccrhyotAeirmacasfhnnobldcusiwpn7onbiarndtiothtniapotdGoiennirnlbsytogiefesdtKshhoiuraAofemnwclttaedhegdoodaeueismngahhhnueahdtnnaugcMmeeegdniaguenbrwsaaliy,hscsti1aoet9enrz8iydeo9mnl)yfz.eoytiumcsnhaedo,cwttthihvseaitt4ym-tfh(uoKetldhaumnimgtuhhateegarnatelilydeyttsilcoayzlas.y,comtz1iyv9emi8ti7yne; AsnS7-.) Gly (N37G) replacement with Asp"' -.) Gly (DlOlG) andTrpa2 + Tyr(W62Y)conversions enwhich TI$* was replaced with Tyr showed at best twice the lytic activity of the wild-type hen enzyme
We report the findings in raphy (Blake et al, 1965)
The emission spectrum of the wild-type eluted a t a n earlier retention time than the corresponding peak of lysozyme is included for comparison
Summary
Trp and Tyr were collected and subjected to amino acid sequence analysis on an Applied Biosystems 473A Protein Sequencer. The emission spectrum of the wild-type eluted a t a n earlier retention time than the corresponding peak of lysozyme is included for comparison. Sequence analysis of this peptide peak clearly showed the replacement of Trp with Tyr a t position 62 of the W62Y mutant enzyme (data not shown). Lysofor the mutantlysozyme was found to be 336 nm, which is at a slightly shorter wavelength than that of the wild-type enzyme (341 nm). When (GICNAC)is~ bound to the wild-type lysozymes at pH 7.5, the fluorescence emission is enhanced zyme was added to 1 ml of a suspension of M. lysodeikticus cells in and its maximum is blue-shifted from 341 to 335 nm. 50 mM sodium phosphate buffer (pH 6.2) or in 0.1 M NaCl and 50 other hand,the quantum yield of the W62Y mutant was found
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