Some Lactobacillus L-lactate dehydrogenases exhibit comparable catalytic activities for pyruvate and oxaloacetate

Abstract

The nonallosteric and allosteric L-lactate dehydrogenases of Lactobacillus pentosus and L. casei, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate K(m) values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the L. pentosus enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate.

FIG. 1

Saturation curves for pyruvate and oxaloacetate for the wild-type and mutant L. pentosus l-LDHs. Reaction velocities for the wild-type (A) and P101N mutant (B) L. pentosus l-LDHs were measured in the presence of the indicated concentrations of pyruvate (open symbols) or oxaloacetate (closed symbols). Dashed and solid lines indicate the calculated saturation curves for pyruvate and oxaloacetate, respectively, by kinetic parameters shown in Table 1.

FIG. 2

Saturation curves for pyruvate and oxaloacetate for L. casei l-LDH at pH 5.0. Reaction velocities were measured in the presence of the indicated concentrations of pyruvate (open symbols) or oxaloacetate (closed symbols), without (circles) or with (squares) 5 mM Fru(1,6)P₂. Dashed and solid lines indicate the calculated saturation curves for pyruvate and oxaloacetate, respectively, by kinetic parameters shown in Table 2.

FIG. 3

Alignment of amino acid sequences of the active-site loops of l-LDHs of bacilli and lactic acid bacteria. LPLDH, L. pentosus (28); LCLDH, L. casei (23); LPLLDH, L. plantarum (9); PALDH, Pediococcus acidilactici (12); SPLDH, Streptococcus mutans (30); STLDH, S. thermophilus (31); LLLDH, Lactococcus lactis (16, 24); BSLDH, B. stearothermophilus (2); BCLDH, B. caldotenax (3); BMLDH, B. megaterium (33); BPLDH, B. psychrosaccharolyticus (32); DFMLDH, dogfish muscle (31). Proline residues conserved in l-LDHs of lactic acid bacteria are shaded.