Mdh 6f5z5y

THE MOBILE LOOP OF GLYOXYSOME MALATE DEHYDROGENASE Alvin Yang University of Richmond

Malate Dehydrogenase ■ An enzyme that catalyzes the oxidation of malate to oxaloacetate using the reduction from NAD+ to NADH.

Malate Dehydrogenase ■ Multimeric enzyme (identical subunits organized as usually either dimer or tetramer) ■ 30-35kDa ■ Each subunit function independently ■ No evidence of cooperativity between catalytic sites ■ Mitochondrial MDH, cytoplasmic MDH, chloroplast MDH, lactate dehydrogenase, glyoxysomal MDH, and so forth

Krebs Cycle

Glyoxysome ■ Consists of a specific group of enzymes enclosed by a single membrane. ■ Function is to destroy hydrogen peroxide (H2O2). ■ Involved in storage lipid mobilization in growing seeds via Glyoxylate cycle. ■ Produce succinate which is converted to sucrose in the cytosol.

Glyoxysome

Glyoxylate cycle

1. Three arginine residues that are responsible for stabilizing malate. 2. Two arginine residues (R102 and R109) are on the mobile loop. 3. R102 and R171 were responsible for the orientation of the substrate at the active site. 4. R102Q mutant enzyme showed lower degree of specificity for the substrate oxaloacetate.

Glyoxysomal Malate Dehydrogenase

Glyoxysomal Malate Dehydrogenase ■ Arrows: Arginine residues

Active Site Mobile Loop of gMDH 1. There are three arginine that facilitate the proper binding of malate to the enzyme’s active site (R124, R130, and R196) 2. The carboxyl group on malate binds to the positively charged arginine residue via electrostatic attraction. 3. Two arginine in close proximity (R124 and R130) are located on the mobile loop. 4. There are two proline residues (P123 and P126) provide the

The two proline residues ■ Two proline residues (P123 and P126) might be involved in the stability of the mobile loop. Proline: ring strain (not flexible).

Hypothesis ■ Mutant in which two proline residues to Glycine residues: -The stability of the mobile will change. ■ The mutant enzyme will exhibit lower protein activity: -The mobile loop is involved in ligand binding.

Overall experiments ■ site directed mutation ■ Protein purification ■ Characterization

-Bradford assay (concentration of the protein) -Circular Dichroism (structural) -Protein Kinetics (function) -MALDI-TOF (mass)

Site-directed mutagenesis (Primer design) ■ gene sequence: 5- gtc cct gca ggt gtt cct cga aaa cca gga atg acg agg gat-3 ■ Forward primer 5-atc cct cgt cat tcc tcc ttt tcg acc aac acc tgc agg gac -3 ■ Reverse primer 5- gtc cct gca ggt gtt ggt cga aaa gga gga atg acg agg gat-3 P123-> G123

P126-> G126

Site-directed mutagenesis 1.Make an isolated plasmid DNA 2.Perform QuikChange: addition of primes we designed. 3.DNA dpn restriction enzyme to destroy the native strand. 4.Transfect E.coli with mutant DNA. 5.Plate the E.coli on ampicillin agarose plates 6.Select colonies that grow

Protein purification

Characterization ■ Protein concentration (Bradford assay) ■ Enzyme kinetics (Km and Vmax) ■ CD ■ MALDI-TOF (Matrix-assisted laser desorption ionization)

Protein concentration ■ Bradford assay -Bradford dye binds to the target protein -bound form: absorption spectrum maximum at 595nm. -using beer’s law to determine the concentration of the protein: A=ebc C=A/eb.

MALDI-TOF

CD spectra

20

0 200

205

210

215

220

225

230

235

240

245

250

-20

-40

Eplliticity

Native

-60

L192W -80

-100

-120

Wavelength (nm)

Double mutant

Enzyme Kinetics

Table 1. Enzymatic kinetics results as varying the concentration of ligands: oxaloacetate (A), and NADH (B). The Vmax, Km and standard deviation were averaged over 5 runs. -Kinetics data was not able to obtain due to low concentration of Pdouble mutant

Conclusions ■ We could not obtain enough concentration of P-double mutant ■ CD data: possible conformational changes (need to rerun) ■ Kinetics: did not obtain the data ■ MALDI: did not obtain the data ■ The possible contamination during the mutagenesis or purification step

Future directions ■ P-double mutant should be remade ■ CD should be retaken. ■ Kinetics: need to run. ■ MALDI: need the information

Acknowledgements ■ Dr. Bell lab -Zach -Zaka -Kelsey -Nikia

Funding source:

References Minarik ,P., Tomaskova, N., Kollarova, M., and Antalik, M.(2002) Malate DehydrogenasesStructure and Function. Gen. Physiol. Biophys, 21, 257-265. Donaldson, R.P., Assadi, M., Karyotou, K., Olcum, T., Qiu, T.(2001) Plant Cells: Peroxisomes and Glyoxysomes, Encyclopedia of life sciences. Goward, C.R., and Nicholls, D.J. (1994) Malate dehydrogenase: A model for structure, evolution, and catalysis, Protein Science, 3, 1883-1888. Images: http://en.wikipedia.org/wiki/Malate_dehydrogenase http ://www.chegg.com/homework-help/lehninger-principles-of-biochemistry-5th-edition-chapte r-16-solutions-9781429277716