Related Experiment Video
Updated: Jun 10, 2026
09:10
Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities
Published on: May 27, 2015
Converting GLX2-1 into an active glyoxalase II
Pattraranee Limphong1, Nicole E Adams, Matthew F Rouhier
1Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
Biochemistry
|August 19, 2010
Summary
Investigating Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) revealed that specific mutations restore catalytic activity. These findings highlight the importance of metal ion coordination and enzyme-substrate interactions for glyoxalase 2 function.
Area of Science:
- Biochemistry
- Enzymology
- Plant Science
Background:
- Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) shares sequence similarity with active GLX2 enzymes but lacks catalytic activity.
- The substrate for GLX2 enzymes is S-lactoylglutathione (SLG).
Purpose of the Study:
- To identify key amino acid residues essential for GLX2 enzyme activity.
- To understand the role of specific residues in metal binding, protein structure, and catalysis.
Main Methods:
- Site-directed mutagenesis of GLX2-1 at positions 219, 246, 248, 325, and 328.
- Overexpression, purification, and characterization of mutant GLX2-1 enzymes.
- Enzyme kinetics, metal analysis, fluorescence spectroscopy, 1H NMR, and EPR spectroscopy.
Main Results:
- Mutations R246H/N248Y partially restored SLG hydrolase activity.
- Further mutations (Q325R/R328K) enhanced catalytic efficiency (kcat) and substrate affinity (Km).
- Metal analysis and spectroscopy indicated proper metal ion binding and the formation of a dinuclear metal center, with Tyr255 being crucial.
Conclusions:
- Active GLX2 enzymes require a correctly positioned metal center.
- Significant non-metal, enzyme-substrate contacts are vital for catalysis.
- Tyrosine 255 plays a critical role in GLX2 enzyme function.
More Related Videos
Related Concept Videos
01:13Outcomes of Glycolysis
Nearly all the energy used by cells comes from the bonds that make up complex organic compounds. These organic compounds are broken down into simpler molecules, such as glucose. As a result, cells extract energy from glucose over many chemical reactions—a process called cellular respiration.
Cellular respiration can occur aerobically (with oxygen) or anaerobically (without oxygen). In the presence of oxygen, cellular respiration starts with glycolysis and continues with pyruvate oxidation, the...
Cellular respiration can occur aerobically (with oxygen) or anaerobically (without oxygen). In the presence of oxygen, cellular respiration starts with glycolysis and continues with pyruvate oxidation, the...
01:15Oxidations of Aldehydes and Ketones to Carboxylic Acids
Oxidation of aldehydes and ketones results in the formation of carboxylic acids. Aldehydes, bearing hydrogen next to the carbonyl group, are easily oxidized compared to ketones. This is because an aldehydic proton can easily be abstracted during oxidation.
Aldehydes readily undergo oxidation in strong oxidizing agents such as potassium permanganate and chromic acid. The oxidation can also be carried out using mild oxidizing agents such as silver oxide. In fact, aldehydes can be easily oxidized...
Aldehydes readily undergo oxidation in strong oxidizing agents such as potassium permanganate and chromic acid. The oxidation can also be carried out using mild oxidizing agents such as silver oxide. In fact, aldehydes can be easily oxidized...
01:20Energy-requiring Steps of Glycolysis
Glucose is the source of nearly all energy used by organisms. The first step of converting glucose into usable energy is called glycolysis. Glycolysis occurs in the cytosol of the cell over two phases: an energy-requiring phase and an energy-releasing phase. Over the first three steps, glucose is converted into different forms and attached to two phosphate groups donated by two ATP molecules, resulting in an unstable sugar. In the next two stages, the unstable sugar splits into two sugar...
01:21Glycolysis: Preparatory Phase
In cellular metabolism (the complete breakdown of glucose to extract energy), glycolysis is the first step. Glycolysis takes place in the cytoplasm of both prokaryotic and eukaryotic cells. Glucose enters heterotrophic cells in two ways. One method is through secondary active transport, where the transport takes place against the glucose concentration gradient. The other mechanism uses a group of integral proteins called GLUT proteins, also known as glucose transporter proteins. These...
01:28Energy-releasing Steps of Glycolysis
Glycolysis is divided into two phases based on whether energy is utilized or released. While the first phase consumes ATP, the second phase produces energy in the form of ATP and NADH. The energy is released over a sequence of reactions that turns G3P into pyruvate. The energy-releasing phase—steps 6-10 of glycolysis—occurs twice, once for each of the two 3-carbon sugars produced during steps 1-5 of the first phase.
The first energy-releasing step—the 6th step of glycolysis —consists of two...
The first energy-releasing step—the 6th step of glycolysis —consists of two...
01:22Reactions of Aldehydes and Ketones: Baeyer–Villiger Oxidation
Baeyer–Villiger oxidation converts aldehydes to carboxylic acids and ketones to esters. The reaction uses peroxy acids or peracids and is often catalyzed by acid. The reaction is named after its pioneers, Adolf von Baeyer and Victor Villiger. The reaction is achieved by a wide range of peracids such as m-chloroperoxybenzoic acid (mCPBA), perbenzoic acid (C6H5COOOH), peracetic acid (CH3COOOH), hydrogen peroxide (H2O2), and tert-butyl hydroperoxide (t-BuOOH).
The carbonyl center is activated by...
The carbonyl center is activated by...