An evaluation of the substrate specificity and asymmetric synthesis potential of the cloned L-lactate dehydrogenase from Bacillusstearothermophilus

1989 ◽  
Vol 67 (6) ◽  
pp. 1065-1070 ◽  
Author(s):  
Daniel Bur ◽  
Marcel A. Luyten ◽  
Hla Wynn ◽  
Louis R. Provencher ◽  
J. Bryan Jones ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Asymmetric Synthesis ◽  
Expression Vector ◽  
Alkyl Chain ◽  
Keto Acid ◽  
Hydroxy Acids ◽  
Thermophilic Enzyme ◽  
Preparative Scale ◽  
Keto Acids ◽  
Branched Chain

The potential utility of the L-lactate dehydrogenase of Bacillusstearothermophilus (BSLDH) for stereospecific, preparative-scale reductions of α-keto acids to (S)-α-hydroxy acids of > 99% ee has been demonstrated. BSLDH is a stable, thermophilic, enzyme whose gene has been cloned into a high-expression vector to assure its plentiful supply. Its specificity for keto acid substrates possessing straight- and branched-chain alkyl, cyclopropyl, or phenyl groups has been evaluated in preparative and kinetic terms, and compared with that of the mammalian pig heart enzyme (PHLDH). The specificities of BSLDH and PHLDH are similar, with branched alkyl-chain keto acids being poor substrates for both enzymes. Keywords: enzymes in organic syntheses, lactate dehydrogenase, asymmetric synthesis.

Transamination of branched-chain keto acids by isolated perfused rat kidney.

1978 ◽  
Vol 235 (1) ◽  
pp. E47
Author(s):  
W E Mitch ◽  
W Chan
Keyword(s):  
Amino Acids ◽  
Rat Kidney ◽  
Branched Chain Amino Acids ◽  
Keto Acid ◽  
Branched Chain Amino Acid ◽  
Keto Acids ◽  
Perfused Rat Kidney ◽  
Branched Chain ◽  
Branched Chain Keto Acids ◽  
Isolated Rat

Isolated rat kidney perfused without substrate released serine, glycine, and taurine, and substantially smaller amounts of other amino acids. When branched-chain keto acids were added, the corresponding amino acids were released at rates amounting to 15-25% of keto acid disappearance. Perfusion with 2 mM alpha-keto-isovalerate or alpha-keto-beta-methylvalerate caused an increased glucose release amounting to 18-23% of keto acid disappearance. The activity of branched-chain amino acid transferase (BATase) was significantly stimulated by perfusion with the analogue of leucine, but not by perfusion with alpha-ketoglutarate, the analogues of valine or isoleucine, or with leucine itself. These findings document that the kidney converts branched-chain keto acids in part to the corresponding amino acids and suggest that the keto analogue of leucine may be involved in the control of renal BATase activity, thereby indirectly regulating the metabolism of branched-chain amino acids.

scholarly journals NET BLOOD EXCHANGE OF BRANCHED-CHAIN AMINO AND α-KETO ACIDS ACROSS THE PORTAL-DRAINED VISCERA AND HINDLIMB OF CATTLE DURING INFUSIONS OF LEUCINE AND INSULIN

1989 ◽  
Vol 69 (1) ◽  
pp. 131-140 ◽  
Author(s):  
R. J. EARLY ◽  
J. R. THOMPSON ◽  
R. J. CHRISTOPHERSON
Keyword(s):  
Amino Acid ◽  
Key Words ◽  
Plasma Glucose ◽  
Whole Blood ◽  
Fold Increase ◽  
Keto Acid ◽  
Branched Chain Amino Acid ◽  
Keto Acids ◽  
Blood Exchange ◽  
Branched Chain

The effects of intra external iliac arterial infusions of leucine (114 μmol h−1 kg0.75) and insulin (0.34 U h−1 kg0.75) into the hindlimb on net whole blood branched-chain amino acid (BCAA), plasma branched-chain α-keto acid (BCKA) and glucose exchange across the hindlimb (HL) and portal-drained viscera (PDV) were investigated in chronically catheterized cattle. Leucine infusions increased (P < 0.05) arterial leucine and α-ketoisocaproate concentrations but did not affect the concentrations of other BCAA, BCKA or glucose. Leucine infusions resulted in a 4-fold increase (P < 0.1) in the net HL removal of leucine and a small increase (P < 0.1) in the net HL release of α-ketoisocaproate. Net whole blood BCAA, plasma BCKA and plasma glucose exchange across the PDV were unaffected by leucine infusions. Insulin infusions decreased (P < 0.1) whole blood leucine, plasma α-ketoisocaproate and plasma glucose concentrations and increased (P < 0.1) the HL extraction of plasma glucose. The HL and PDV extraction of whole blood BCAA and plasma BCKA were unaffected by insulin infusions. The data suggest that cattle are less sensitive to the effects of leucine and insulin on tissue BCAA catabolism compared to nonruminant species. Key words: Branched-chain amino acid, branched-chain α-keto acid, leucine, insulin, cattle

BIOCHEMICAL STUDIES ON A VARIANT OF BRANCHED CHAIN KETOACIDURIA IN A 19-YEAR-OLD FEMALE

PEDIATRICS ◽  
1971 ◽  
Vol 48 (5) ◽  
pp. 795-801
Author(s):  
Milton H. Fischer ◽  
Theo Gerritsen
Keyword(s):  
Newborn Infants ◽  
Good Health ◽  
Branched Chain Amino Acids ◽  
Keto Acid ◽  
Fatal Disease ◽  
Maple Syrup ◽  
Keto Acids ◽  
Urine Disease ◽  
Branched Chain ◽  
Peripheral Leukocytes

Branched chain ketoaciduria (BCKA) or maple syrup urine disease is a rare, serious, and usually early fatal disease found in newborn infants. The finding of 233 mg of branched chain α-keto acids in the urine of a young adult was therefore surprising. The patient was physically in good health and had a full scale IQ of 76. The excretion of branched chain amino acids and branched chain α-keto acids could be increased by feeding a diet high in protein. Branched chain α- keto acid dehydrogenase activities in the peripheral leukocytes and in skin fibroblasts were found to be decreased to only a very small fraction of control values and were comparable to the levels found in a case of classic BCKA.

BLOOD BRANCHED-CHAIN AMINO AND α-KETO ACID CONCENTRATIONS AND NET EXCHANGE ACROSS THE PORTAL-DRAINED VISCERA AND HINDLIMB OF FED AND FASTED RUMINANTS

1987 ◽  
Vol 67 (4) ◽  
pp. 1011-1020 ◽  
Author(s):  
RICHARD J. EARLY ◽  
JAMES R. THOMPSON ◽  
ROBERT J. CHRISTOPHERSON ◽  
GARY W. SEDGWICK
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hind Limb ◽  
Venous Blood ◽  
Branched Chain Amino Acids ◽  
Keto Acid ◽  
Branched Chain Amino Acid ◽  
Keto Acids ◽  
Branched Chain ◽  
Cattle And Sheep

In the first of two experiments, whole blood branched-chain amino acid (BCAA) and plasma branched-chain α-keto acid (BCKA) concentrations in jugular venous blood were determined in cattle and sheep before and during a 6-d fast. In cattle, concentrations of valine, isoleucine, α-ketoisovalerate (KIV) and α-ketomethylvalerate (KMV) remained unchanged whereas leucine and α-ketoisocaproate (KTC) increased (P < 0.05) during fasting. In sheep, only KIV and KMV remained unchanged whereas BCAA and KIC increased (P < 0.05) during fasting. In a second experiment on cattle chronically catheterized to measure BCAA and BCKA exchange across the portal-drained viscera (PDV) and hindlimb (HL), the PDV added and the HL removed BCAA from the blood of fed cattle. The opposite exchange occurred after a 6-d fast. Releases of BCKA from the PDV and HL in both fed and fasted states were small compared to BCAA exchanges. The data suggest that blood BCAA but not BCKA concentrations may respond differently to starvation in sheep versus cattle and that in cattle the PDV and HL do not release appreciable amounts of BCKA relative to the net movements of the BCAA. Key words: Portal-drained viscera, hind limb, branched-chain amino acids, branched-chain α-keto acids, fasting, ruminants

BRANCHED-CHAIN ALPHA-KETO ACID EXCHANGE ACROSS THE PORTAL-DRAINED VISCERA AND HINDLIMB OF FED AND FASTED STEERS

1984 ◽  
Vol 64 (5) ◽  
pp. 276-277 ◽  
Author(s):  
R. J. EARLY ◽  
J. R. THOMPSON ◽  
R. J. CHRISTOPHERSON ◽  
G. W. SEDGWICK
Keyword(s):  
Key Words ◽  
Keto Acid ◽  
Keto Acids ◽  
Branched Chain ◽  
Net Release ◽  
Acid Exchange

Fasting (6 day) steers increased (P < 0.05) arterial alpha-ketoisocaproate concentration and decreased (P < 0.05) the net release of alpha-ketoisovalerate from the portal-drained viscera. The net release of all three branched-chain alpha-keto acids from the hindlimb was decreased (P < 0.05) after fasting. Key words: Branched-chain, alpha-keto acids, steers

scholarly journals Branched-Chain α-Keto Acid Catabolism via the Gene Products of the bkd Operon in Enterococcus faecalis: a New, Secreted Metabolite Serving as a Temporary Redox Sink

2000 ◽  
Vol 182 (11) ◽  
pp. 3239-3246 ◽  
Author(s):  
Donald E. Ward ◽  
Coen C. van der Weijden ◽  
Marthinus J. van der Merwe ◽  
Hans V. Westerhoff ◽  
Al Claiborne ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Enterococcus Faecalis ◽  
Physiological Role ◽  
Reduced Form ◽  
Gene Products ◽  
Keto Acid ◽  
Anaerobic Conditions ◽  
Keto Acids ◽  
Branched Chain ◽  
Responsive Element

ABSTRACT Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain α-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkdgene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain α-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the α-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of α-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD+ from the NADH produced by the branched-chain α-keto acid dehydrogenase complex was required for complete conversion of α-ketoisocaproate. Interestingly, during the conversion of the branched-chain α-keto acids an intermediate was always detected extracellularly. With α-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1,1-dihydroxy-4-methyl-2-pentanone. This reduced form of α-ketoisocaproic acid was found to serve as a temporary redox sink.

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