Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy

J. M. Goldberg, J. Zheng, Hua Deng, Y. Q. Chen, Robert Callender, J. F. Kirsch

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The azomethine (Schiff base) linkage between the ε-amino group of active- site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'- phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (λ(max) = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H2 18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H2 18O. A molecular orbital explanation for the anomalous red shift in the near-UV spectrum of the Y225F-PLP complex is proposed. In addition, the O3' atom of PLP is found to simultaneously accept hydrogen bonds from Tyr-225 and Lys- 258 in the protonated internal aldimine form of AATase.

Original languageEnglish (US)
Pages (from-to)8092-8097
Number of pages6
JournalBiochemistry
Volume32
Issue number32
StatePublished - 1993
Externally publishedYes

Fingerprint

Pyridoxal Phosphate
Raman Spectrum Analysis
Aspartate Aminotransferases
Phenylalanine
Isotopes
Escherichia coli
Tyrosine
Spectroscopy
Schiff Bases
Lysine
Hydrogen
Hydrogen bonds
Apoenzymes
Holoenzymes
Valine
Molecular orbitals
Enzymes
Aldehydes
Raman spectroscopy
Raman scattering

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{de30578bb7b54bf693c5a695fdd7bc7b,
title = "Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy",
abstract = "The azomethine (Schiff base) linkage between the ε-amino group of active- site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'- phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (λ(max) = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H2 18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H2 18O. A molecular orbital explanation for the anomalous red shift in the near-UV spectrum of the Y225F-PLP complex is proposed. In addition, the O3' atom of PLP is found to simultaneously accept hydrogen bonds from Tyr-225 and Lys- 258 in the protonated internal aldimine form of AATase.",
author = "Goldberg, {J. M.} and J. Zheng and Hua Deng and Chen, {Y. Q.} and Robert Callender and Kirsch, {J. F.}",
year = "1993",
language = "English (US)",
volume = "32",
pages = "8092--8097",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "32",

}

TY - JOUR

T1 - Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy

AU - Goldberg, J. M.

AU - Zheng, J.

AU - Deng, Hua

AU - Chen, Y. Q.

AU - Callender, Robert

AU - Kirsch, J. F.

PY - 1993

Y1 - 1993

N2 - The azomethine (Schiff base) linkage between the ε-amino group of active- site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'- phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (λ(max) = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H2 18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H2 18O. A molecular orbital explanation for the anomalous red shift in the near-UV spectrum of the Y225F-PLP complex is proposed. In addition, the O3' atom of PLP is found to simultaneously accept hydrogen bonds from Tyr-225 and Lys- 258 in the protonated internal aldimine form of AATase.

AB - The azomethine (Schiff base) linkage between the ε-amino group of active- site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'- phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (λ(max) = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H2 18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H2 18O. A molecular orbital explanation for the anomalous red shift in the near-UV spectrum of the Y225F-PLP complex is proposed. In addition, the O3' atom of PLP is found to simultaneously accept hydrogen bonds from Tyr-225 and Lys- 258 in the protonated internal aldimine form of AATase.

UR - http://www.scopus.com/inward/record.url?scp=0027218352&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027218352&partnerID=8YFLogxK

M3 - Article

VL - 32

SP - 8092

EP - 8097

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 32

ER -