Design of a specific phenyllactate dehydrogenase by peptide loop exchange on the Bacillus stearothermophilus lactate dehydrogenase framework

H M WILKS, K M MORETON, D J HALSALL, K W HART, R D SESSIONS, A R CLARKE, J J HOLBROOK, Richard B Sessions

Research output: Contribution to journalArticle (Academic Journal)peer-review

Abstract

Restriction sites were introduced into the gene for Bacillus stearothermophilus lactate dehydrogenase which enabled a region of the gene to be excised which coded for a mobile surface loop of polypeptide (residues 98-110) which normally seals the active site vacuole from bulk solvent and is a major determinant of substrate specificity. Oligonucleotide-overlap extension (using the polymerase chain reaction) was used to obtain double-stranded DNA regions which coded for different length and sequence loops and which also contained the same restriction sites. The variable length and sequence loops were inserted into the cut gene and used to synthesize hydroxyacid dehydrogenases with altered substrate specificities. Loops which were longer and shorter than the original were made. The substrate specificities of enzymes with these new loops were considerably altered. For many poor enzyme-substrate pairs, the effect of fructose 1,6-bisphosphate on the steady-state kinetic parameters suggested that the substrate was mainly bound in a nonproductive mode, With one longer loop construction (BL1), activity with pyruvate was reduced one-million-fold but activity with phenylpyruvate was largely unaltered. A switch in specificity (k(cat)/K(M)) of 390 000-fold was achieved. The 1700:1 selectivity of enzyme BL1 for phenylpyruvate over pyruvate is that required in a phenyllactate dehydrogenase to be used in monitoring phenylpyruvate in the urine of patients with phenylketonuria consuming an apparently phenylalanine-free diet.

Translated title of the contributionDesign of a specific phenyllactate dehydrogenase by peptide loop exchange on the Bacillus stearothermophilus lactate dehydrogenase framework
Original languageEnglish
Pages (from-to)7802-7806
Number of pages5
JournalBiochemistry
Volume31
Issue number34
Publication statusPublished - 1 Sep 1992

Fingerprint

Dive into the research topics of 'Design of a specific phenyllactate dehydrogenase by peptide loop exchange on the <i>Bacillus stearothermophilus</i> lactate dehydrogenase framework'. Together they form a unique fingerprint.

Cite this