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Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

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Title Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase
 
Creator Jala, Venkatakrishna Rao
Ambili, M
Prakash, V
Rao, N Appaji
Savithri, H S
 
Subject Active site geometry
Distal interactions
Geminal diamine
Glycine-external aldimine
Pyridoxal-5’-phosphate
Serine hydroxymethyltransferase (SHMT)
Site-directed mutagenesis
 
Description 226-237
The crystal structure of human liver cytosolic
recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and
Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of
Arg262 (corresponding to Arg263 in hcSHMT) to 'A' in sheep liver cytosolic SHMT
(scSHMT) resulted in a 5-fold increase in Km for L-Ser
and a 5-fold decrease

in kcat compared to
scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal
diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow
studies showed that the rate constant for the formation of glycine-geminal
diamine for R262A SHMT was also decreased. The rate of reaction, concentration
of spectral intermediates, fluorescence excitation maximum of glycine geminal
diamine and interaction with methoxyamine were altered in R262A SHMT. Although
Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73
for interaction with PLP, by forked H-bonding with the carbonyl groups of main
chain residues, Asn71 and Lys72 of the other subunit of the tight dimer.
Mutation of Arg262 to Ala
and the consequent alteration in orientation of PLP leads to decreased
catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one
of the carboxylate oxygens of the amino acid substrate, which also interacts
with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT
resulted in disruption of these interactions, whereas replacement with Ala
(S53A) only weakened the substrate interactions. There was a 10-fold increase
in Km and 20-fold decrease in catalytic activity
efficiency for S52C

SHMT, whereas S52A SHMT retained 20% of the
activity without change in Km for serine. These
results suggest that S52 affects substrate binding and catalysis.
 
Date 2015-01-19T05:39:08Z
2015-01-19T05:39:08Z
2003-08
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/30399
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.40(4) [August 2003]