1gg6: Difference between revisions

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CRYSTAL STUCTURE OF GAMMA CHYMOTRYPSIN WITH N-ACETYL-PHENYLALANINE TRIFLUOROMETHYL KETONE BOUND AT THE ACTIVE SITE

OverviewOverview

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.

About this StructureAbout this Structure

1GG6 is a Protein complex structure of sequences from Bos taurus with , , and as ligands. Active as Chymotrypsin, with EC number 3.4.21.1 Full crystallographic information is available from OCA.

ReferenceReference

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin., Neidhart D, Wei Y, Cassidy C, Lin J, Cleland WW, Frey PA, Biochemistry. 2001 Feb 27;40(8):2439-47. PMID:11327865

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-[[Image:1gg6.gif|left|200px]]<br /><applet load="1gg6" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1gg6.gif|left|200px]]<br /><applet load="1gg6" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1gg6, resolution 1.4&Aring;" />
 '''CRYSTAL STUCTURE OF GAMMA CHYMOTRYPSIN WITH N-ACETYL-PHENYLALANINE TRIFLUOROMETHYL KETONE BOUND AT THE ACTIVE SITE'''<br />
 ==Overview==
-The structures of the hemiketal adducts of Ser 195 in chymotrypsin with, N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and, N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined, to 1.4-1.5 A by X-ray crystallography. The structures confirm those, previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and, Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings, between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A, resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between, His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium, isotope effects. The X-ray crystal structure of the hemiacetal adduct, between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal, (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to, the AcLF-CF3 adduct, except for the presence of two epimeric adducts in, the R- and S-configurations at the hemiacetal carbons. In the, (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion, site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is, not protonated at Nepsilon2, and there is no LBHB at pH &gt;7.0. Because, addition of AcLF-CHO to chymotrypsin neither releases nor takes up a, proton from solution, it is concluded that the hemiacetal oxygen of the, chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the, oxyanion site. The protonation states of the hemiacetal and His 57 are, explained by the high basicity of the hemiacetal oxygen (pK(a) &gt; 13.5), relative to that of His 57. The 13C NMR signal for the adduct of, AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal, between pH 7 and 13. At pH &lt;7.0, His 57 in the AcLF-CHO-hemiacetal complex, of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a, transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the, active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy, barrier of 6-7 kcal x mol(-1) against ionizations that change the, electrostatic charge at the active site. However, ionizations of neutral, His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by, LBHB formation, suggesting that stabilization by the LBHB overcomes the, barrier to ionization. On the basis of the hydration constant for, AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold, larger than the comparable value for AcLF-CF3.
+The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH &gt;7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) &gt; 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH &lt;7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.
 ==About this Structure==
-GG6 is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with SO4, APF, APL and EDO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Chymotrypsin Chymotrypsin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.1 3.4.21.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1GG6 OCA].
+GG6 is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=SO4:'>SO4</scene>, <scene name='pdbligand=APF:'>APF</scene>, <scene name='pdbligand=APL:'>APL</scene> and <scene name='pdbligand=EDO:'>EDO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Chymotrypsin Chymotrypsin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.1 3.4.21.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1GG6 OCA].
 ==Reference==
@@ Line 15: / Line 15: @@
 [[Category: Protein complex]]
 [[Category: Cassidy, C.]]
-[[Category: Cleland, W.W.]]
+[[Category: Cleland, W W.]]
-[[Category: Frey, P.A.]]
+[[Category: Frey, P A.]]
 [[Category: Lin, J.]]
 [[Category: Neidhart, D.]]
@@ Line 26: / Line 26: @@
 [[Category: chymotrypsin]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 16:01:36 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:49:41 2008''