Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 12 November

© R. Paselk 2008
 
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Glycolysis, cont.

10) Pyruvate Kinase: PEP to Pyruvate

structural diagram of the reaction catalyzed by pyruvate kinase

Here we have an attack by ADP:

structural diagram of the reaction mechanism for Pyruvate Kinase

The resulting enol then spontaneously tautomerizes to pyruvate.

PK is a regulatory enzyme in some tissues. There are three isozymes:

PK completes the reactions of Glycolysis. However, for Glycolysis to proceed NAD+ needs to be regenerated. For aerobic tissues this is done via the Kreb's TCA Cycle. Later we will look at this process for aerobic cells.

Lactate DH is used to regenerate NAD+ in anaerobic tissue in mammals, and takes Pyruvate to Lactate:

 

structural diagram of the reaction catalyzed by Lactate dehydrogenase

Again the NAD+ abstracts a Hydride ion in the reverse reaction:

structural diagram of the reaction mechanism for the conversion of lactate to pyruvate by Lactate DH

while a general base aids the formation of the carbonyl carbon, and a positive charge draws electron charge up to the carboxyl group and aids the removal of the hydride ion.

Lactate DH also has isozymes. It is a tetramer of two types of monomers, H & M. Can thus have 5 possible isomers: H4, H3M, H2M2, HM3, & M4, with one active site per monomer.The kinetic properties of the pure monomer LD isozymes are given in the Table:

Michaelis Constant (KM)
  H M
Pyruvate 1.4 x 10-4 5.2 x 10-4
Lactate 9 x 10-3 2.5 x 10-2
Pyruvate Inhibition? yes no
The properties of the H(eart) monomer, which predominates in aerobic tissues can be rationalized as better adapted to the aerobic environment. (Heart uses lactate from the serum as a fuel, but doesn't want to lose pyruvate produced in glycolysis to lactate production. The properties of the mixed isozymes will be intermediate.

Gluconeogenesis

In order to provide glucose for vital functions such as the metabolism of RBC's and the CNS during periods of fasting (greater than about 8 hrs after food absorption in humans), the body needs a way to synthesis glucose from precursors such as pyruvate and amino acids. This process is referred to as gluconeogenesis. It occurs in the liver and in kidney. Most of Glycolysis can be used in this process since most glycolytic enzymes are operating at equilibrium. However three irreversible enzymes must be bypassed in gluconeogenesis vs. glycolysis: Hexokinase, Phosphofructokinase, and Pyruvate kinase. Phosphofructokinase, and/or hexokinase must also be bypassed in converting other hexoses to glucose.

Let's begin with pyruvate. How is pyruvate converted to PEP without using the pyruvate kinase reaction? Formally, pyruvate is first converted to oxaloacetate, which is in turn converted to PEP. In the first reaction of this process Pyruvate carboxylase adds carbon dioxide to pyruvate with the expenditure of one ATP equivalent of energy. Biotin, a carboxyl-group transfer cofactor in animals, is required by this enzyme:

overall chemical reaction of pyruvate and carbon dioxide to oxaloacetate

The reaction takes place in two parts on two different sub-sites on the enzyme. In the first part biotin attacks bicarbonate with a simultaneous attack/hydrolysis by bicarbonate on ATP, resulting in the release of ADP and inorganic phosphate (note the coupling by the enzyme of independent processes in this reaction):

structural diagram of the reaction mechanism for the carboxylation of biotin by Pyruvate carboxylase

Note that the 14 Angstrom arm of biocytin allows biotin to move between the two sites, in this case carrying the activated carboxyl group. In the second site a pyruvate carbanion then attacks the activated carboxyl group, regenerating the biotin cofactor and releasing oxaloacetate:

structural diagram of the reaction mechanism for the transfer of the activated carboxyl group from biotin to pyruvate catalyzed by Pyruvate Carboxylase

Pyruvate carboxylase is followed by the Phosphoenolpyruvate carboxykinase (PEPCK) reaction. In this reaction oxaloacetate is decarboxylated with a simultaneous phosphorylation by GTP to give GDP:

structural diagram of the reaction mechanism for PEP Carboxykinase

 

In eukaryotes the transformation of Pyruvate to Phosphoenol pyruvate (PEP) is further complicated by the fact that oxaloacetate is generated from pyruvate and TCA Cycle intermediates only in the mitochondria, while PEP is converted to glucose in the cytosol. And oxaloacetate cannot cross the mitochondrial membrane efficiently (it is present at concentrations way below the KM of the carrier, so it must be converted into malate or aspartate in order to cross as summarized in the diagram: gluconeogenesis in the liver.


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Last modified 12 November 2008