Pedro Mendes wrote, in response to my prior posting: "What we have to do in
order to apply MCA to these systems is to look
at the complex of enzymes that are involved in channelling as ONE
ENTITY. Then if we change the concentration of this entity the overall
rate of its (complex) reaction will change linearly. There is no
conflict with the summation theorem if we view it in this way."
I THINK I UNDERSTAND THE POINT PEDRO IS MAKING. BUT, AS CAN BE DEDUCED FROM
OTHER PARTS OF HIS POSTING, IT DOES NOT SEEM TO LEAD TO INFORMATIVE
EXPERIMENTS. AS ALL READERS KNOW, THE ENTRIES IN THE SUM ARE THE FRACTIONAL
CHANGE IN THE FLUX RESULTING FROM THE FRACTIONAL CHANGE IN EACH ENZYME
CONCENTRATION (WHILE HOLDING ALL OTHER ENZYME CONCENTRATIONS CONSTANT.
WHILE IT IS POSSIBLE TO CONCEIVE OF THE COMPLEX AS A SINGLE ENTITY, THERE
IS NO EXPERIMENTAL WAY THAT I AM AWARE OF TO VARY THE CONCENTRATION OF THE
COMPLEX SYSTEMATICALLY OVER A WIDE RANGE OF VALUES NOR OF SATISFYING THE
CONDITION IMPOSED BY TAKING THE PARTIAL DERIVATIVE OF [COMPLEX]; NAMELY, TO
LET IT VARY WHILE HOLDING [E-1] AND [E-2] CONSTANT.
BUT THE THEORY IN ITS PRESENT STATE OF DEVELOPMENT DOES SUGGEST
EXPERIMENTS. CONSIDER THE SIMPLE CASE OF A TWO ENZYME "PATHWAY" IN WHICH
[E-1] = [E-2], AS AN EXAMPLE. IMAGINE THAT CHANNELLING OCCURS IN THIS
PATHWAY. THE EXPERIMENTER CAN VARY [E-T] ([E-T] = [E-1] + [E-2]) OVER A
WIDE RANGE. SINCE THE CHANNELLED PATHWAY INCLUDES TERMS WHICH ARE FIRST
ORDER IN [E-1] (THE BINDING OF SUBSTRATE TO E-1) AND OTHER STEPS WHICH ARE
SECOND ORDER (COMPLEX FORMATION), THE PATHWAY FLUX CAN NOT BE REPRESENTED
BY A HOMOGENEOUS FUNCTION OF FIRST ORDER AND THUS, AS I UNDERSTAND IT,
THERE IS NO NECESSITY FOR THE SUM OF THE CONTROL COEFFICIENTS TO BE UNITY.
SO, IF FLUX THROUGH A PATHWAY PROCEEDS VIA A "LEAKY CHANNEL" (that is, part
channelled, part not), THEN THE SUM OF COEFFICIENTS (WHICH WE EVALUATE AS
THE SLOPE OF THE PLOT OF LN FLUX VS LN [E-T]) SHOULD DEPEND ON [E-T]. (SEE
FIG 5 IN CHAPTER BY KHOLODENKO, CASCANTE & WESTERHOFF IN CHANNELLING IN
INTERMEDIARY METABOLISM, ED. AGIUS & SHERRATT, 1997 PORTLAND PRESS). WHILE
WE DO HAVE TO HAVE VALUES FOR RATE CONSTANTS TO EVALUATE THE FRACTION
CHANNELLED (J-sub chan / J), IT SEEMS TO ME THAT WE CAN PROPERLY INFER THAT
SOME PART OF THE FLUX IS CHANNELED ON THE BASIS OF THE VARIATION OF THE SUM
OF THE CONTROL COEFFICIENTS AS A FUNCTION OF [E-T].
WHAT I HAD IN MIND IN MY EARLIER POSTING WAS THAT EMPHASIZING THAT THE SUM
OF THE CONTROL COEFFICIENTS FOR A PATHWAY EQUALS ONE (IF YOU DEFINE THE
"STEPS" CORRECTLY) OBSCURES THE POSSIBILITY OF DOING EXPERIMENTS SUCH AS
THAT DESCRIBED ABOVE WHICH ARE MOTIVATED BY MCA AND WHICH PROVIDE A MEANS
OF DETERMINING WHETHER CHANNELING PLAYS A ROLE IN A PATHWAY
AT THE BEGINNING OF HIS LETTER, PEDRO WROTE A SUMMARY OF HIS VIEWS
(SLIGHTLY EDITED BELOW):
>-1. I think that metabolite channelling is a very important phenomenon
> which is widespread in various cell types.
>-2. MCA can be applied to pathways that have metabolite channelling
> as long as we realize that the whole enzyme complex is now one single
> entity or if we consider the elementary reactions of the whole
> complex.
>-3. The above means that we can and should apply MCA to these pathways
PEDRO ADDED PARENTHETICALLY "(though I thought Danny was aware of it (MY
VIEWS)!)".
I AM AWARE THAT PEDRO THINKS CHANNELLING IS AN IMPORTANT PHENOMENON. I DID
NOT MEAN TO SUGGEST OTHERWISE. IF ANYTHING I WROTE DID SUGGEST THAT PEDRO
WAS UNINTERESTED IN CHANNELLING, I APOLOGIZE. IN FACT, PEDRO, WITH
CONSIDERABLE PATIENCE, INTRODUCED ME TO MCA AS AN APPROACH TO STUDYING
CHANNELLING AT THE LAST GORDON CONFERENCE. MY SUBSTANTIVE COMMENTS ARE
DIRECTED TOWARD HIS POINT 2 ABOVE. IT SEEMS TO ME THAT IT SETS THE LIMITS
TOO NARROWLY ON WHAT CAN BE DONE EXPERIMENTALLY.
______________
> We must
>however also realize that channelling is (functionally) the fusion of
>two (or more) steps into one. In some cases this has even happened on
>the genome,
AS AN ASIDE UNRELATED TO MY TEXT ABOVE, WE HAVE EXPERIENCE WITH ONE CASE IN
WHICH TWO STEPS ARE FUSED INTO ONE. (WE WERE NOT AWARE OF THE CHANNELING
LITERATURE AT THE TIME.) THE OUTCOME SURPRISED US THEN AND STILL DOES. IN
SOYBEAN NODULE BACTEROIDS, (THE SITE OF N2 FIXATION WITHIN NODULES FORMED
AS A RESULT OF THE INFECTION OF SOYBEAN ROOTS WITH BRADYRHIZOBIUM
JAPONICUM), PROLINE IS REDUCED via
E-1 E-2
proline ------------- to P5C ------------- to glutamate
where E-1 = proline dehydrogenase and E-2 = P5C reductase. (arrows don't
reproduce in email)
E-1 AND E-2 ARE TRANSCRIBED FROM A POLYCISTRONIC MESSAGE. AN in vitro
ASSAY FOR P5C INVOLVES THE FORMATION OF AN ADDUCT BETWEEN P5C AND
ORTHO-AMINOBENZOIC ACID (OAB). IF P5C WERE 100% CHANNELED AS A RESULT OF
THE FUSION OF E-1 AND E-2, THEN IT WOULD BE IMPOSSIBLE TO TRAP P5C WITH
OAB. YET UP TO 75% OF IT was TRAPPED (Shearer, et al. Analytical Biochem
1992, 203: 191-200). THUS, PROTEIN/PROTEIN INTERACTION WOULD SEEM TO BE A
NECESSARY, BUT NOT SUFFICIENT, CONDITION FOR CHANNELLING.
With apologies for the length of this posting,
Danny Kohl
