Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

被引:31
作者
Jagath, JR
Sharma, B
Bhaskar, B
Datta, A
Rao, NA
Savithri, HS
机构
[1] INDIAN INST SCI, DEPT BIOCHEM, BANGALORE 560012, KARNATAKA, INDIA
[2] JAWAHARLAL NEHRU UNIV, SCH LIFE SCI, NEW DELHI 110067, INDIA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1997年 / 247卷 / 01期
关键词
sheep cytosolic serine hydroxymethyltransferase; N-terminal deletion mutant; C-terminal deletion mutant; overexpression; subunit assembly;
D O I
10.1111/j.1432-1033.1997.00372.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using sis amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6) SHMT and des-(A1-W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-/A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 degrees C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52 degrees C) compared to rSHMT (56 degrees C) and des-(A1-K6)-SHMT (55 degrees C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2+/-0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8+/-0.1 M) and des-(A1 -K6)-SHMT (1.7+/-0.1 M). The apoenzyme of des-/A1-K6)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (approximate to 75% and approximate to 65%, respectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively des-(A1-W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity refined correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.
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收藏
页码:372 / 379
页数:8
相关论文
共 37 条
[1]  
ACHARYA JK, 1992, J BIOL CHEM, V267, P19066
[2]  
ALEXANDER DC, 1987, METHOD ENZYMOL, V154, P41
[3]   3-DIMENSIONAL STRUCTURE OF TYROSINE PHENOL-LYASE [J].
ANTSON, AA ;
DEMIDKINA, TV ;
GOLLNICK, P ;
DAUTER, Z ;
VONTERSCH, RL ;
LONG, J ;
BEREZHNOY, SN ;
PHILLIPS, RS ;
HARUTYUNYAN, EH ;
WILSON, KS .
BIOCHEMISTRY, 1993, 32 (16) :4195-4206
[4]   MODE OF INTERACTION OF AMINOOXY COMPOUNDS WITH SHEEP LIVER SERINE HYDROXYMETHYLTRANSFERASE [J].
BASKARAN, N ;
PRAKASH, V ;
SAVITHRI, HS ;
RADHAKRISHNAN, AN ;
RAO, NA .
BIOCHEMISTRY, 1989, 28 (25) :9613-9617
[5]   MECHANISM OF INTERACTION OF O-AMINO-D-SERINE WITH SHEEP LIVER SERINE HYDROXYMETHYLTRANSFERASE [J].
BASKARAN, N ;
PRAKASH, V ;
RAO, AGA ;
RADHAKRISHNAN, AN ;
SAVITHRI, HS ;
RAO, NA .
BIOCHEMISTRY, 1989, 28 (25) :9607-9612
[6]   INTERACTIONS OF L-SERINE AT THE ACTIVE-SITE OF SERINE HYDROXYMETHYLTRANSFERASES - INDUCTION OF THERMAL-STABILITY [J].
BHASKAR, B ;
PRAKASH, V ;
SAVITHRI, HS ;
RAO, NA .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1994, 1209 (01) :40-50
[7]   INTERCONVERSION OF SERINE AND GLYCINE - PARTICIPATION OF PYRIDOXAL PHOSPHATE [J].
BLAKLEY, RL .
BIOCHEMICAL JOURNAL, 1955, 61 (02) :315-323
[8]   Interaction of sheep liver apo-serine hydroxymethyltransferase with pyridoxal-5'-phosphate: A physicochemical, kinetic, and thermodynamic study [J].
Brahatheeswaran, B ;
Prakash, V ;
Savithri, HS ;
Rao, NA .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1996, 330 (02) :363-372
[9]  
FUKUMOTO Y, 1991, J BIOL CHEM, V266, P4187
[10]   CALCULATION OF PROTEIN EXTINCTION COEFFICIENTS FROM AMINO-ACID SEQUENCE DATA [J].
GILL, SC ;
VONHIPPEL, PH .
ANALYTICAL BIOCHEMISTRY, 1989, 182 (02) :319-326