The active sites of fructose 6-phosphate,2-kinase: Fructose-2,6- bisphosphatase from rat testis. Roles of ASP-128, THR-52, THR-130, ASN-73, and TYR-197

Kosaku Uyeda, Xiao Li Wang, Hiroyuki Mizuguchi, Yang Li, Cu Nguyen, Charles A. Hasemann

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Abstract

To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate,2-kinase:fructose- 2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr- 197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in V(max) and a significant increase in K(m) values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high K(m) for both substrates and an 800-fold decreased V(max). Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in K(Fru) (6-P) with only a small increase in K(ATP) and small changes in V(max). Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased V(max). A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with K(m)= 0.64 μM, and V(max) = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The K(d) values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.

Original languageEnglish (US)
Pages (from-to)7867-7872
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number12
DOIs
StatePublished - 1997

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Phosphofructokinase-2
Testis
Rats
Catalytic Domain
Enzymes
Adenosine Triphosphate
Phosphotransferases
Substrates
Nucleophiles
Catalysis
Mutation
Viperidae
Substitution reactions
Stabilization
Fluorescence
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

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The active sites of fructose 6-phosphate,2-kinase : Fructose-2,6- bisphosphatase from rat testis. Roles of ASP-128, THR-52, THR-130, ASN-73, and TYR-197. / Uyeda, Kosaku; Wang, Xiao Li; Mizuguchi, Hiroyuki; Li, Yang; Nguyen, Cu; Hasemann, Charles A.

In: Journal of Biological Chemistry, Vol. 272, No. 12, 1997, p. 7867-7872.

Research output: Contribution to journalArticle

Uyeda, Kosaku ; Wang, Xiao Li ; Mizuguchi, Hiroyuki ; Li, Yang ; Nguyen, Cu ; Hasemann, Charles A. / The active sites of fructose 6-phosphate,2-kinase : Fructose-2,6- bisphosphatase from rat testis. Roles of ASP-128, THR-52, THR-130, ASN-73, and TYR-197. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 12. pp. 7867-7872.
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abstract = "To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate,2-kinase:fructose- 2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr- 197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in V(max) and a significant increase in K(m) values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high K(m) for both substrates and an 800-fold decreased V(max). Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in K(Fru) (6-P) with only a small increase in K(ATP) and small changes in V(max). Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased V(max). A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with K(m)= 0.64 μM, and V(max) = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The K(d) values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.",
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AU - Uyeda, Kosaku

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AU - Nguyen, Cu

AU - Hasemann, Charles A.

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N2 - To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate,2-kinase:fructose- 2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr- 197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in V(max) and a significant increase in K(m) values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high K(m) for both substrates and an 800-fold decreased V(max). Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in K(Fru) (6-P) with only a small increase in K(ATP) and small changes in V(max). Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased V(max). A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with K(m)= 0.64 μM, and V(max) = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The K(d) values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.

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