Jens J. Birktoft

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.

[8] Glutamyl endopeptidases

Publisher Summary This chapter highlights glutamyl endopeptidases, with emphasis on GluV8 from S. aureus (strain V8), GluSGP from Streptomyces griseus, and GluBL from Bacillus licheniformis. A large number of glutamic acid-containing peptides can be employed in assaying the enzymatic activities of glutamyl endopeptidases. These include benzyloxycarbonyl-Leu-Leu-Glu-β-naphthylamide, carbobenzyloxycarbonyl-Phe-Leu-Glu- p -nitroanilide, and o -aminobenzoyl-(anthraniloyl)-Ala-Ala-Glu-Val-Tyr(NO 2 )-Asp-OH. Depending on the substrate employed, different techniques are used to monitor enzyme activities: (1) nitroanilide substrate hydrolysis is determined spectrophotometrically at 410 nm, (2) substrates containing the anthraniloyl and 3-nitrotyrosine groups are assayed by monitoring the fluorescence emission at 420 nm on excitation at 320 nm, and (3) the release of β-naphthylamide is followed fluorometrically. The optimal pH is dependent on the specific enzymes used, varying between pH 7.3 and 9.0, and also on the substrate employed. All three Glu-specific proteases display a pronounced preference for Glu-Xaa bonds versus Asp-Xaa bonds. For most endopeptidases the subsite interactions have a noticeable influence on the efficiency of substrate hydrolysis.

Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.

Crystallographic studies of glutamate dehydrogenase. II. Preliminary crystal data for the tuna liver enzyme.

X-ray examination of large crystals of glutamate dehydrogenase prepared from yellowfin tuna liver indicate that the crystals belong to space group I23 (or I213) with a = b = c = 228·4 A. The unit cell contains 72 subunits (12 hexameric molecules) of molecular weight about 55,500, and there is one half-molecule, i.e. three subunits in the asymmetric unit. The X-ray diffraction data indicate that the crystalline tuna liver glutamate dehydrogenase appears more suitable for structural analysis than the previously obtained crystals of the rat liver enzyme (Birktoft et al., 1979).

Preliminary X-ray diffraction analysis of a crystallizable mutant of malate dehydrogenase from the thermophile Thermus flavus.

Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies. The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A. The asymmetric unit consists of one homodimer of molecular mass 67,000 Da. The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected.