scholarly journals Binding energy and specificity in the catalytic mechanism of yeast aldose reductases

1999 ◽  
Vol 344 (1) ◽  
pp. 101-107 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Peter MAYR ◽  
Philipp HADWIGER ◽  
Arnold E. STüTZ
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Catalytic Mechanism ◽  
Relative Proportion ◽  
Binding Constants ◽  
Stabilization Energy ◽  
Binary Complex ◽  
Hydrogen Bonding Interactions ◽  
Transition State Stabilization ◽  
Hydroxy Groups

Derivatives of D-xylose and D-glucose, in which the hydroxy groups at C-5, and C-5 and C-6 were replaced by fluorine, hydrogen and azide, were synthesized and used as substrates of the NAD(P)H-dependent aldehyde reduction catalysed by aldose reductases isolated from the yeasts Candida tenuis, C. intermedia and Cryptococcus flavus. Steady-state kinetic analysis showed that, in comparison with the parent aldoses, the derivatives were reduced with up to 3000-fold increased catalytic efficiencies (kcat/Km), reflecting apparent substrate binding constants (Km) decreased to as little as 1/250 and, for D-glucose derivatives, up to 5.5-fold increased maximum initial rates (kcat). The effects on Km mirror the relative proportion of free aldehyde that is available in aqueous solution for binding to the binary complex enzyme-NAD(P)H. The effects on kcat reflect non-productive binding of the pyranose ring of sugars; this occurs preferentially with the NADPH-dependent enzymes. No transition-state stabilization energy seems to be derived from hydrogen-bonding interactions between enzyme-NAD(P)H and positions C-5 and C-6 of the aldose. In contrast, unfavourable interactions with the C-6 group are used together with non-productive binding to bring about specificity (6-10 kJ/mol) in a series of D-aldoses and to prevent the reaction with poor substrates such as D-glucose. Azide introduced at C-5 or C-6 destabilizes the transition state of reduction of the corresponding hydrogen-substituted aldoses by approx. 4-9 kJ/mol. The total transition state stabilization energy derived from hydrogen bonds between hydroxy groups of the substrate and enzyme-NAD(P)H is similar for all yeast aldose reductases (yALRs), at approx. 12-17 kJ/mol. Three out of four yALRs manage on only hydrophobic enzyme-substrate interactions to achieve optimal kcat, whereas the NAD(P)H-dependent enzyme from C. intermedia requires additional, probably hydrogen-bonding, interactions with the substrate for efficient turnover.

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

Aziridine Scaffolds for the Detection and Quantification of Hydrogen-Bonding Interactions through Transition-State Stabilization

2010 ◽  
Vol 123 (3) ◽  
pp. 767-770
Author(s):  
Luciana Giordano ◽  
Cam T. Hoang ◽  
Michael Shipman ◽  
James H. R. Tucker ◽  
Tiffany R. Walsh
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Hydrogen Bonding Interactions ◽  
Transition State Stabilization ◽  
Detection And Quantification

Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain

Biochemistry ◽  
1991 ◽  
Vol 30 (22) ◽  
pp. 5531-5538 ◽  
Author(s):  
Robert Menard ◽  
Henry E. Khouri ◽  
Celine Plouffe ◽  
Pierre Laflamme ◽  
Robert Dupras ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Catalytic Mechanism ◽  
Side Chain ◽  
Hydrogen Bonding Interactions

Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase

Biochemistry ◽  
1990 ◽  
Vol 29 (8) ◽  
pp. 2075-2080 ◽  
Author(s):  
Ann Lewendon ◽  
Iain A. Murray ◽  
William V. Shaw ◽  
Michael R. Gibbs ◽  
Andrew G. W. Leslie
Keyword(s):  
Transition State ◽  
Catalytic Mechanism ◽  
Chloramphenicol Acetyltransferase ◽  
Transition State Stabilization

scholarly journals Crystal structure of (2-benzyloxypyrimidin-5-yl)boronic acid

2014 ◽  
Vol 70 (12) ◽  
pp. o1259-o1260
Author(s):  
Krzysztof Durka ◽  
Tomasz Kliś ◽  
Janusz Serwatowski
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Boronic Acid ◽  
Acid Group ◽  
Stacking Interactions ◽  
Aromatic Rings ◽  
Compound C ◽  
Hydrogen Bonding Interactions ◽  
Hydroxy Groups ◽  
Centrosymmetric Dimers

The boronic acid group in the title compound, C11H11BN2O3, adopts asyn–anticonformation and is almost coplanar with the aromatic rings , making a dihedralangle of 3.8 (2)°. In the crystal, adjacent molecules are linkedviapairs of O—H...O interactions, forming centrosymmetric dimers with anR22(8) motif, which have recently been shown to be energetically very favorable (Durkaet al., 2012, 2014). The hydroxy groups in ananticonformation are engaged in lateral hydrogen-bonding interactions with N atoms from neighbouring molecules, leading to the formation of chains along [001]. O...B [3.136 (2) Å] and C(π)...B [3.393 (2) Å] stacking interactions in turn link parallel chains of centrosymmetric dimers into layers parallel to (010).

scholarly journals Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases

2003 ◽  
Vol 373 (2) ◽  
pp. 319-326 ◽  
Author(s):  
Kathryn L. KAVANAGH ◽  
Mario KLIMACEK ◽  
Bernd NIDETZKY ◽  
David K. WILSON
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Hydrogen Bonds ◽  
Metabolic Pathway ◽  
Xylose Reductase ◽  
High Flux ◽  
R Factor ◽  
Hydrogen Bonding Interactions ◽  
Dual Specificity ◽  
Hydroxy Groups

Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD+ to 1.80 Å resolution (where 1 Å=0.1 nm) with a crystallographic R-factor of 18.3%. A comparison of the NAD+- and the previously determined NADP+-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2′-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn276, which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu227 on a short rigid helix, which makes hydrogen bonds to both the 2′- and 3′-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3′-endo conformation rather than the 2′-endo conformation seen in the NADP+-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

Sign in / Sign up

Export Citation Format

Share Document