Enhancement of coenzyme binding by a single point mutation at the coenzyme binding domain of E. coli lactaldehyde dehydrogenase.
Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) share some structural and kinetic properties. One difference is that PAD can use NAD+ and NADP+, whereas ALD only uses NAD+. An acidic residue has been involved in the exclusion of NADP+ from the active site in pyridine nucleotide-dependent dehydrogenases. However, other factors may participate in NADP+ exclusion. In the present work, analysis of the sequence of the region involved in coenzyme binding showed that residue F180 of ALD might participate in coenzyme specificity. Interestingly, F180T mutation rendered an enzyme (ALD-F180T) with the ability to use NADP+. This enzyme showed an activity of 0.87 micromol/(min * mg) and K(m) for NADP+ of 78 microM. Furthermore, ALD-F180T exhibited a 16-fold increase in the V(m) /K(m) ratio with NAD+ as the coenzyme, from 12.8 to 211. This increase in catalytic efficiency was due to a diminution in K(m) for NAD+ from 47 to 7 microM and a higher V(m) from 0.51 to 1.48 micromol/(min * mg). In addition, an increased K(d) for NADH from 175 (wild-type) to 460 microM (mutant) indicates a faster product release and possibly a change in the rate-limiting step. For wild-type ALD it is described that the rate-limiting step is shared between deacylation and coenzyme dissociation. In contrast, in the present report the rate-limiting step in ALD-F180T was determined to be exclusively deacylation. In conclusion, residue F180 participates in the exclusion of NADP+ from the coenzyme binding site and disturbs the binding of NAD+.
Rodríguez-Zavala
José Salud
JS
Departamento de Bioquímica, Instituto Nacional de Cardiología "Ignacio Chávez," Tlalpan D.F. 14080, Mexico. rodjos@cardiologia.org.mx
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013696:Temperature
D029968:Escherichia coli Proteins; D009243:NAD; D009249:NADP; D000445:Aldehyde Oxidoreductases; C021493:lactaldehyde dehydrogenase