Subunit interface mutants of rabbit muscle aldolase form active dimers.


Abstract

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. Study holds ProTherm entries: 10967, 10968, 10969, 10970, 10971, 10972, 10973, 10974, 10975, 10976, 10977, 10978, 10979 Extra Details: additive : NaN3(0.02 %),transition1 aldolase; quaternary structure; site-directed mutagenesis;,subunit interface; thermolability

Submission Details

ID: JVKHnp2E3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Beernink PT;Tolan DR,Protein Sci. (1994) Subunit interface mutants of rabbit muscle aldolase form active dimers. PMID:7833800
Additional Information

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
1ALD 1991-05-05T00:00:00+0000 2.0 ACTIVITY AND SPECIFICITY OF HUMAN ALDOLASES
2ALD 1998-10-21T00:00:00+0000 2.1 HUMAN MUSCLE ALDOLASE
4ALD 1998-07-26T00:00:00+0000 2.8 HUMAN MUSCLE FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE COMPLEXED WITH FRUCTOSE 1,6-BISPHOSPHATE
5KY6 2016-07-21T00:00:00+0000 1.94 Human muscle fructose-1,6-bisphosphate aldolase
6XMH 2020-06-30T00:00:00+0000 1.95 Human aldolase A wild type
6XML 2020-06-30T00:00:00+0000 1.88 Human aldolase A I98C
6XMM 2020-06-30T00:00:00+0000 2.11 Human aldolase A I98S
6XMO 2020-06-30T00:00:00+0000 2.6 Human aldolase A I98F
1ADO 1996-12-02T00:00:00+0000 1.9 FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE FROM RABBIT MUSCLE
1EWD 2000-04-25T00:00:00+0000 2.46 FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE FROM RABBIT MUSCLE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Fructose-bisphosphate aldolase A P00883 ALDOA_RABIT
98.4 Fructose-bisphosphate aldolase A P04075 ALDOA_HUMAN
98.1 Fructose-bisphosphate aldolase A Q5NVR5 ALDOA_PONAB
98.1 Fructose-bisphosphate aldolase A P05064 ALDOA_MOUSE
97.8 Fructose-bisphosphate aldolase A A5A6I5 ALDOA_PANTR
97.3 Fructose-bisphosphate aldolase A P05065 ALDOA_RAT