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:1287659Additional Information
Study Summary
Structure view and single mutant data analysis
Study data
| 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 |
|---|---|---|---|
| 5MQ5 | 2017-11-15 | 1.6 | A protease-resistant N24S Escherichia coli Asparaginase mutant with outstanding stability and enhanced anti-leukaemic activity |
| 6PAC | 2019-09-04 | 1.6 | E. coli L-asparaginase II in complex with L-Asp at pH 5.6 |
| 6PA3 | 2019-09-04 | 1.65 | E. coli L-asparaginase II double mutant (T89V,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 |
| 6V23 | 2020-05-20 | 1.75 | Ligand-free L-aspraginase II from E. coli (EcAII) |
| 6V25 | 2020-05-20 | 1.78 | Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp |
| 6V26 | 2020-05-20 | 1.8 | Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp |
| 6PAA | 2019-09-04 | 1.85 | E. coli L-asparaginase II mutant (T12V) in complex with L-Asp at pH 5.5 |
| 6NX8 | 2019-08-07 | 1.85 | ECAII(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 6.2 |
| 6PA4 | 2019-09-04 | 1.85 | E. coli L-asparaginase II double mutant (T89V,K162T) in complex with L-Asp at pH 7.0 |
| 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 |
| 6V2G | 2020-05-20 | 1.9 | Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp. Covalent acyl-enzyme intermediate and tetrahedral intermediate. |
| 6PA8 | 2019-09-04 | 1.9 | ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 7.0 |
| 6V24 | 2020-05-20 | 1.9 | Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp. Covalent acyl-enzyme intermediate. |
| 6NXA | 2019-08-07 | 1.93 | ECAII(D90T,K162T) MUTANT AT PH 7 |
| 6V28 | 2020-05-20 | 1.95 | Complex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asp |
| 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(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 7 |
| 6V29 | 2020-05-20 | 2.0 | Complex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asp |
| 6V2A | 2020-05-20 | 2.0 | Complex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asn |
| 6PA5 | 2019-09-04 | 2.0 | ECAII(T89V,K162T) MUTANT IN COMPLEX WITH L-ASN AT PH 8.3 IN SPACE GROUP P2(1) |
| 6V2C | 2020-05-20 | 2.0 | Complex of mutant (K162M) of E. coli L-asparaginase II with L-Asp. Covalent acyl-enzyme intermediate and tetrahedral intermediate |
| 6V2B | 2020-05-20 | 2.05 | Complex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asn |
| 6UOH | 2020-10-21 | 2.1 | Asparaginase II from Escherichia coli |
| 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(D90T,K162T) MUTANT IN COMPLEX WITH CITRATE AT PH 5 |
| 6NX7 | 2019-08-07 | 2.15 | ECAII(D90T,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 |
| 6UOG | 2020-10-21 | 2.29 | Asparaginase II from Escherichia coli |
| 6V27 | 2020-05-20 | 2.3 | Complex of double mutant (T89V,K162T) of E. coli L-asparaginase II with L-Asp |
| 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 (ELSPAR) |
| 6UOD | 2020-10-21 | 2.4 | Asparaginase II from Escherichia coli |
| 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 |