Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis.

Abstract

Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important. Study holds ProTherm entries: 9876, 9877, 9878, 9879, 9880 Extra Details: asparaginase II; Escherichia coli ; mutagenesis; serine; threonine

Submission Details

ID: ZkrD2cKo3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:39 p.m.

Version: 1

Publication Details
Derst C;Henseling J;Röhm KH,Protein Eng. (1992) Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis. PMID:1287659
Additional Information

Study Summary

Number of data points 5
Proteins L-asparaginase 2 ; L-asparaginase 2
Unique complexes 5
Assays/Quantities/Protocols Experimental Assay: dG_H2O
Libraries Mutations for sequence LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
6PAC 2019-09-04 1.6 E. coli L-asparaginase II in complex with L-Asp at pH 5.6
5MQ5 2017-11-15 1.6 A protease-resistant N24S Escherichia coli Asparaginase mutant with outstanding stability and enhanced anti-leukaemic activity
6PA3 2019-09-04 1.65 E. coli L-asparaginase II double mutant (T12V,K162T) in complex with L-Asn at pH 7.0
6PAB 2019-09-04 1.73 E. coli L-asparaginase II in complex with L-Asp at pH 7.0
6NXB 2019-08-07 1.75 ECAII IN COMPLEX WITH CITRATE AT PH 7
6NX8 2019-08-07 1.85 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 6.2
6PA4 2019-09-04 1.85 E. coli L-asparaginase II double mutant (T12V,K162T) in complex with L-Asp at pH 7.0
6PAA 2019-09-04 1.85 E. coli L-asparaginase II mutant (T12V) in complex with L-Asp at pH 5.5
6PA9 2019-09-04 1.88 E. coli L-asparaginase II mutant (T12V) in complex with L-Asn at pH 7.0
6PA2 2019-09-04 1.9 E. coli L-asparaginase II mutant (K162M) in complex with L-Asp at pH 5.6
6PA8 2019-09-04 1.9 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 7.0
6NXA 2019-08-07 1.93 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 7
1NNS 2003-03-11 1.95 L-asparaginase of E. coli in C2 space group and 1.95 A resolution
6NX9 2019-08-07 1.97 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 7
6PA5 2019-09-04 2.0 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 8.3 IN SPACE GROUP P2(1)
6PA6 2019-09-04 2.12 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 8.3 in space group C2
6NX6 2019-08-07 2.15 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 5
6NX7 2019-08-07 2.15 ECAII(T89V,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 5.6
4ECA 1997-06-16 2.2 ASPARAGINASE FROM E. COLI, MUTANT T89V WITH COVALENTLY BOUND ASPARTATE
1JAZ 2003-09-09 2.27 Crystal Structure of Monoclinic Form of D90E Mutant of Escherichia coli Asparaginase II
1JJA 2003-09-09 2.3 CRYSTAL STRUCTURE OF ORTHORHOMBIC FORM OF D90E MUTANT OF ESCHERICHIA COLI L-ASPARAGINASE II
3ECA 1993-10-31 2.4 CRYSTAL STRUCTURE OF ESCHERICHIA COLI L-ASPARAGINASE, AN ENZYME USED IN CANCER THERAPY
6EOK 2018-10-31 2.5 Crystal structure of E. coli L-asparaginase II
1HO3 2001-03-07 2.5 CRYSTAL STRUCTURE ANALYSIS OF E. COLI L-ASPARAGINASE II (Y25F MUTANT)
1IHD 2003-09-09 2.65 Crystal Structure of Trigonal Form of D90E Mutant of Escherichia coli Asparaginase II

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 L-asparaginase 2 P00805 ASPG2_ECOLI